Inducible immunomodulating tumor-homing bacteria

Abstract

A recombinant tumor-homing bacterium having constitutive prokaryotic expression cassettes comprising two or more homologous cancer associated antigens, each of the respective homologous cancer associated antigens associated with a transport signal from a distinct transport system, a regulating expression cassette encoding a regulator and an aspirin-inducible prokaryotic expression cassette encoding a set of immunomodulator fusion peptides operably linked to an inducible promoter, each of said immunomodulator fusion peptides comprising a heterologous immunomodulator associated with a secretion signal, said heterologous immunomodulator selected from a list consisting of: LIGHT, GMCSF, SIRP alpha, and IL18.

Description

INDUCIBLE IMMUNOMODULATING TUMOR-HOMING BACTERIACROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is based on and claims benefit of priority of U.S. Provisional Patent Application No. 63 / 556,139, filed Feb. 21, 2024, Israel Patent Application No. 311017, filed Feb. 21, 2024, as well as P.C.T Patent Application No. PCT / IB2024 / 058038, filed Aug. 21, 2024, the contents of which are incorporated herein by reference in their entireties.TECHNICAL FIELD

[0002] The present disclosure in some embodiments thereof, relate to recombinant bacteria, related vaccines, and methods of cancer treatment using recombinant tumor-homing bacterium genetically modified for inducible heterologous immunomodulators. More specifically, the present invention, in some embodiments thereof, relate to recombinant tumor-homing bacterium, related vaccines, and methods of cancer treatment using recombinant tumor-homing bacterium genetically modified to constitutively produce cancer-associated antigens and inducible heterologous immunomodulators.REFERENCE TO A SEQUENCE LISTING

[0003] The application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on filed Feb. 21, 2025, is named BAC00590000 and is 87 kilobytes in size.BACKGROUND

[0004] Certain tumors are challenging to treat based on conventional methods. Tumors have been known to employ advanced mechanisms for evading host innate immune responses in many ways such as downregulating antigen presentation on MHC class I molecules or expressing suppressive molecules to resist cytotoxic T Lymphoctye recognition and attack, or recruiting fibroblasts for establishing a physical barrier to prevent immune infiltration. Although recombinant tumor-homing bacteria engineered to deliver tumor-local treatment have been in development for decades, to date there are no approved bacteria with this capability for use in the treatment of cancer.

[0005] Recombinant tumor-homing bacteria must meet multiple criteria for successful tumorlocal treatment of cancer, and specifically tumor-local delivery of efficacious biologically activepeptides. Specific and sufficient tumor colonization by bacteria in a host is necessary and requires overcoming the antibacterial properties of human serum and the harsh conditions of the tumor microenvironment. Additionally, Gram-negative bacteria engineered to express Eukaryotic peptides, must enable these peptides to cross multiple barriers, including the inner and outer lipid membranes and the periplasmic space, to be effective. In addition, functional Eukaryotic peptides delivered by bacteria to a tumor, may be dependent on post-translational modifications or more complex secondary and tertiary protein folding, dependent on physical and chemical properties of the environment in which it folds.

[0006] Moreover, when the peptide is an immunomodulator, targeted tumor-specific secretion with minimal systemic impact and controlled timing is also desired. Further, even if delivery to a tumor of the biologically active form of a peptide is achieved, it may not be delivered in a relevant dose for efficacious treatment either because of the limited amount of inducer that can be administered without causing negative clinical manifestation or alternatively, due to limitations of the bacteria in the area of tumor-local secretion.

[0007] Furthermore, when targeting tumor-specific peptide secretion, achieving a balance between effective immunomodulation and avoiding an immunosuppressive effect is crucial. For example, a constant and high level of immunomodulation may have an immune suppressive effect. Additionally, not all immunomodulator payload combinations offer synergistic benefits.

[0008] Additionally, in the case of induced secretion of a bacterial strain within a host, the presence of an inducer may introduce an additional level of complexity, especially when the inducer is known to have an anti-inflammatory effect.

[0009] Thus, it is advantageous to develop tumor-homing bacteria capable of controlled, doserelevant peptide delivery for effective tumor treatment. In view of the challenges outlined herein, there remains a need for engineering tumor-homing bacteria and methods for the efficacious treatment a tumor to abrogate immune evasion by the tumor.SUMMARY

[0010] The present disclosure relates to a genetically modified tumor-homing bacterium engineered for inducible expression and tumor-local delivery of biologically active immunomodulators and its use in cancer treatment. More particularly, the present disclosure relates to a genetically modified tumor-homing bacterium engineered for constitutive multimodal transport of cancer-associated antigens as well as inducible expression and tumor-local delivery of biologically active heterologous immunomodulators.

[0011] This summary is provided to introduce a selection of concepts in a simplified form that are further described below in the detailed description. This summary is not intended to identify key features or essential features of the claimed subject matter, nor is it intended to be used as an aid in determining the scope of the claimed subject matter.

[0012] According to an aspect of the present disclosure, a recombinant tumor-homing bacterium is provided. Embodiments consistent with the present disclosure provide for novel tumor-homing bacterium genetically modified for immune system priming and activation as well as time- controlled tumor-local delivery of biologically active immunomodulators in efficacious doses such that systemic side effects are limited while tumor reduction is achieved.

[0013] According to an aspect of the present disclosure, a tumor-homing bacterium genetically modified for inducible tumor-local delivery of multiple heterologous immunomodulators is provided. The tumor-homing bacterium is an attenuated Gram-negative bacterium comprising three prokaryotic expression cassettes: one or more constitutive prokaryotic expression cassettes comprising two or more homologous cancer associated antigens, each of the respective homologous cancer associated antigens associated with a transport signal from a distinct transport system; a regulating expression cassette encoding a regulator; and one or more aspirininducible prokaryotic expression cassettes encoding a set of immunomodulator fusion peptides operably linked to an inducible promoter, each of said immunomodulator fusion peptides comprising a heterologous immunomodulator associated with a secretion signal, said heterologous immunomodulator selected from a list consisting of: LIGHT, GMCSF, SIRP alpha, and IL18.

[0014] According to other aspects of the present disclosure, the tumor-homing bacterium may include one or more of the following features. The aspirin-inducible prokaryotic expression cassettes may encode three peptides selected from the list of: LIGHT, SIRP alpha, GMCSF and IL 18. A ratio of the number of inducible prokaryotic expression cassettes to the regulating expression cassette may be 2: 1 or more. Upon induction, the immunomodulator may be secreted in a biologically active form. The tumor-homing bacterium may have no deactivating mutation in an outer membrane protein; and each of the respective heterologous immunomodulators may be linked to a secretion signal from a Type III secretion system or an inner membrane secretion system. The heterologous immunomodulator may have a cysteine content of more than 1 %, orbetween 1 - 4%, and may be linked to a secretion signal from a sec or tat secretion system. The polynucleotide encoding the LIGHT peptide may be inserted at the adl locus. The distinct transport signals may be a Type III and a Type V secretion signal. The distinct transport signals may each be of a surface display signal, a Type III secretion signal and a Type V secretion signal. The inducible prokaryotic expression cassette may be inserted at the adl, ttrA, or Stm3120 locus. The regulating expression cassette may be in trans position relative to the inducible prokaryotic expression cassettes. The regulating expression cassette encoding a regulator may be either: inserted at the aadA locus rendering a deactivating mutation in the aadA gene; or in a trans position relative to the inducible prokaryotic expression cassette and wherein the inducible promoter is induced by a salicylic acid or derivative thereof. The inducible prokaryotic expression cassette may be a polycistronic vector comprising three or more polynucleotide sequences selected from the list of: LIGHT, SIRP alpha, GMCSF and IL 18. The homologous cancer associated antigens may be homologous neoantigens. The tumor-homing bacterium may be selected from the list consisting of: Salmonella spp., Yersinia spp., Bordetella spp., Escherichia coli, Shigella spp., Burkholderia mallei, Burkholderia pseudomallei and Pseudomonas aeruginosa. The bacterium may be selected from a genus being a Salmonella and / or a Pseudomonas. The bacterium may be a species being Salmonella Typhimurium. The expression cassettes may be chromosomally integrated.

[0015] According to another aspect of the present disclosure, a tumor-homing bacterium, genetically modified for inducible tumor-local delivery of multiple heterologous immunomodulators is provided. The tumor-homing bacterium is an attenuated bacteria having no deactivating mutations in an outer membrane and having a total of two prokaryotic expression cassettes comprising: one or more constitutive prokaryotic expression cassettes comprising two or more homologous cancer associated antigen (e.g., homologous neoantigen polynucleotides), each associated with a transport signal from a distinct transport system, and an inducible prokaryotic expression cassettes encoding an immunomodulator fusion peptide comprising a heterologous immunomodulator linked to a secretion signal, wherein either: the secretion signal is an inner membrane secretion signal or a Type III secretion signal; or further comprising a prokaryotic expression cassette comprising a regulator positioned upstream or in a trans configuration relative to the inducible prokaryotic expression cassette.

[0016] According to other aspects of the present disclosure, the tumor-homing bacterium may include one or more of the following features. The inducer may be a small molecule selectedfrom the list consisting of L-arabinose, IPTG, a salicylic acid, acetylsalicylic acid or derivative thereof. The inducible prokaryotic expression cassette may be an aspirin-inducible prokaryotic expression cassette.

[0017] According to another aspect of the present disclosure, a tumor-homing bacterium, genetically modified for inducible tumor-local delivery of multiple heterologous immunomodulators is provided. The tumor-homing bacterium is an attenuated bacterium having no deactivating mutations in an outer membrane and having a total of three prokaryotic expression cassettes comprising: one or more constitutive prokaryotic expression cassettes comprising two or more homologous cancer associated antigen (e.g., homologous neoantigen polynucleotides), each associated with a transport signal from a distinct transport system, an aspirin inducible prokaryotic expression cassette encoding an immunomodulator fusion peptide comprising a secretion signal linked to a heterologous immunomodulator; and a regulating expression cassette inserted at a aadA locus or positioned in a trans configuration relative to the aspirin-inducible expression cassette.

[0018] According to another aspect of the present disclosure, a vaccine comprising a bacterium according to any one of the previously described tumor-homing bacteria and one or more pharmaceutically acceptable carriers or excipients is provided.

[0019] According to another aspect of the present disclosure, a use of the vaccine in the treatment of cancer in a subject in need thereof is provided. The use comprises: administering to the subject an effective amount of the vaccine; upon tumor-colonization of the bacteria in a cancer tumor and / or a reduction in a systemic bacterial load in a subject; administering to the subject a salicylate, salicylic acid or derivative thereof; and administering to the subject an immune checkpoint modulator.

[0020] According to other aspects of the present disclosure, the use may include one or more of the following features. Administering an inducer may be a single-time systemic administration. Administering an inducer may be a periodic administration. Administering an effective amount of the vaccine may be by parenteral administration. The cancer may be selected from the group consisting of breast, melanoma, colorectal cancer, lung cancer, gastric cancer, pancreatic cancer, ovarian cancer, bone cancer and brain cancer.

[0021] According to another aspect of the present disclosure, a method of treating a cancer in a subject is provided. The method comprises: administering to a blood stream of a subject in need thereof, a tumor-homing bacterium genetically modified for constitutive multi-model secretionof two homologous cancer associated antigens and inducible expression of a set of heterologous immunomodulators comprising three or more peptides from the following: SIRP alpha, GMCSF, IL 18 and LIGHT; upon tumor-colonization of the bacteria in a cancer tumor and / or a reduction in a systemic bacterial load, administering an inducer; and administering an immune checkpoint modulator.

[0022] According to other aspects of the present disclosure, the method may include one or more of the following features. The two homologous cancer associated antigens may be two homologous neoantigens. Administering the tumor-homing bacterium to a blood stream of a subject may comprise intravenous or intra-tumoral administration. Administering to a blood stream of a subject may comprise intravenous administration. Multi-model secretion of homologous cancer associated antigens may comprise secretion by a Type HI and a Type V secretion system. Further to the multi-model secretion, a homologous neoantigen may be displayed on the cell wall surface. The set of heterologous immunomodulators may comprise all four of the following: SIRP alpha, GMCSF, IL 18 and LIGHT. The tumor-homing bacterium may be a Gram-negative bacterium.BRIEF DESCRIPTION OF THE FIGURES

[0023] Unless otherwise defined, all technical and / or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice of embodiments of the invention, exemplary methods and / or materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting.

[0024] Some embodiments of the invention are herein described by way of example only, with reference to the accompanying drawings. With specific reference now to the drawings in detail, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of embodiments of the invention. In this regard, the description taken with the drawings makes apparent to those skilled in the art how embodiments of the invention may be practiced.

[0025] FIG. 1A and FIG. IB illustrates an aspect of the subject matter in accordance with one embodiment of the recombinant bacterium at the time of administration and upon tumor-homing and induction of immunomodulator expression.

[0026] FIG. 2 illustrates a pSal / SalR system.

[0027] FIG. 3 A illustrates an aspect of the subject matter in accordance with one embodiment of the Salmonella Typhimurium genome and its modifications.

[0028] FIG. 3B illustrates an aspect of the subject matter in accordance with one embodiment of the Salmonella Typhimurium genome and its modifications.

[0029] FIG. 4 illustrates multiple types of bacterial secretion systems.

[0030] FIG. 5 illustrates an aspect of the subject matter in accordance with one embodiment, wherein the kinetics of the aspirin-mediated induction with a salR-pSal system in mice harboring aspirin-inducible luciferase is demonstrated.

[0031] FIG. 6 illustrates an aspect of the subject matter in accordance with one embodiment wherein a recombinant tumor-homing bacterium, where improved protein expression results from the trans configuration of the salR regulator relative to the pSal promoter in a bacteria genome .

[0032] FIG. 7 illustrates functional induction of IL 18 HEK blue cells by the supernatant of a recombinant tumor-homing bacterium genetically modified to express and secrete IL 18, in accordance with one embodiment.

[0033] FIG. 8 illustrates functional induction and secretion of LIGHT secreted by a recombinant tumor-homing bacterium in accordance with one embodiment.

[0034] FIG. 9 illustrates functional induction of GM-CSF reporter cells following incubation with the supernatant of bacteria expressing and secreting GM-CSF by a recombinant tumorhoming bacterium in accordance with one embodiment.

[0035] FIG. 10A illustrates the binding of bacterial-expressed SIRPa by a recombinant tumorhoming bacterium in accordance with one embodiment.

[0036] FIG. 10B illustrates the detection of SIRPa protein in the secreted fraction from a recombinant tumor-homing bacterium in accordance with one embodiment.

[0037] FIG. 11 illustrates insertion of multiple modifications and expression of all inserted coding sequences in a recombinant tumor-homing bacterium, in accordance with one embodiment.

[0038] FIG. 12 presents graphs of IL18-mediated immune profile changes in mice tumors and tumor draining lymph nodes (LN) for a tumor-homing bacterium genetically modified with an aspirin-inducible IL 18 peptide, in accordance with one embodiment.

[0039] FIG. 13 presents graphs of LIGHT-mediated immune profile changes in mice tumors and tumor draining lymph nodes (LN) for a tumor-homing bacterium genetically modified with an aspirin-inducible LIGHT peptide, in accordance with one embodiment.

[0040] FIG. 14 presents graphs of GM-CSF-mediated immune profile changes in mice tumors and tumor draining lymph nodes (LN) for a tumor-homing bacterium genetically modified with an aspirin-inducible GM-CSF peptide, in accordance with one embodiment.

[0041] FIG. 15 presents graphs of SIRPa-mediated immune profile changes in mice tumors and tumor draining lymph nodes (LN) for a tumor-homing bacterium genetically modified with an aspirin-inducible SIRPa peptide, in accordance with one embodiment.

[0042] FIG. 16 is a graph demonstrating the average size of tumors (bars) and distribution of tumor sizes in mice for the different immunomodulator treatments as measured on day 17 following injection of a tumor-homing bacterium in accordance with one embodiment.

[0043] FIG. 17 illustrates the efficacy, as measured by tumor volume reduction in mm3in mice, of various tumor-homing bacteria in accordance with embodiments of the present disclosure.TERMS AND DEFINITIONS

[0044] In order that the disclosure may be more readily understood, the following terms used through the application will be first defined. These definitions should be read in the context of the disclosure and understood by a person of ordinary skill in the art. Additional definitions may be dispersed throughout the detailed description.

[0045] Terms like "comprising," "having," "containing," and "including," are all intended to convey a similar meaning. They do not imply an exhaustive list of items or limit the subsequent items to only those mentioned. Furthermore, the singular articles "a," "an," and "the" are applicable to plural cases unless the context specifically indicates otherwise. It should also be noted that relational terms such as "first" and "second" are employed solely to distinguish between different entities or operations, without suggesting any inherent relationship or sequence.

[0046] As used herein, unless specifically stated otherwise, the term “or” encompasses all possible combinations, except where infeasible. For example, if it is stated that a component caninclude A or B, then, unless specifically stated otherwise or infeasible, the component can include A or B, or A and B. As a second example, if it is stated that a component can include at least one of A, B, or C, then, unless specifically stated otherwise or infeasible, the component can include A, B, or C, or A and B, or A and C, or B and C, or A, B, and C.

[0047] Throughout this application, various embodiments of this invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

[0048] Whenever a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integral) within the indicated range. The phrases “ranging / ranges between” a first indicate number and a second indicate number and “ranging / ranges from” a first indicate number “to” a second indicate number are used herein interchangeably and are meant to include the first and second indicated numbers and all the fractional and integral numerals therebetween.

[0049] This disclosure employs open-ended permissive language, indicating for example, that some embodiments “may” employ, involve, or include specific features. The use of the term “may” and other open-ended terminology is intended to indicate that although not every embodiment may employ the specific disclosed feature, at least one embodiment employs the specific disclosed feature.

[0050] As used herein, genetically modified or recombinant are used interchangeably, and, in the context of the present disclosure, refer to a bacteria or bacterium whose genetic material has been altered using genetic engineering techniques such that it has been genetically transformed with a polynucleotide construct like a vector. The recombinant bacterium, which is preferably a Gram negative bacterium, is genetically modified through transformation, transduction or conjugation, and is preferably a recombinant Gram-negative bacterium genetically modified through transformation, transduction or conjugation with multiple polynucleotide molecules to express multiple heterologous immunomodulators and cancer associated antigens (such as neoantigens) for therapeutic purposes.

[0051] As used herein, the terms “polypeptide”, “peptide”, “protein”, “polypeptidic” and “peptide” are used interchangeably to designate a series of amino acid residues connected to each other by peptide bonds between the alpha-amino and carboxy groups of adjacent residues. Preferred are proteins which have an amino acid sequence comprising at least 10 amino acids, more preferably at least 20 amino acids.

[0052] As used herein, a heterologous peptide or protein, or specifically a heterologous immunomodulator or heterologous neoantigen as used herein, is understood to refer to a gene or encoding sequence which codes for a protein or a fragment thereof which originates from a source different from the bacteria into which it is introduced and has been introduced into the Gramnegative bacterium or Gram-negative bacterial strain by genetic transformation, transduction or conjugation. The heterologous protein or a fragment thereof can engineered into the bacterial chromosome or engineered in an extra-chromosomal genetic element of the Gram-negative bacterium. Usually, the heterologous protein or a fragment thereof is of animal origin including human origin. Preferably the heterologous protein or a fragment thereof is a human protein or a fragment thereof.

[0053] As used herein, heterologous immunomodulator (referred to as immunomodulator) refers to a heterologous protein or a fragment thereof or a polynucleotide encoding a heterologous protein or a fragment thereof. It may include naturally occurring proteins or a fragment thereof and also includes artificially engineered proteins or a fragment thereof. Artificially engineered proteins or a fragment thereof are variants or functionally active fragments of the heterologous protein. By “variants or functionally active fragments thereof’ in relation to the heterologous protein of the present invention is meant that the fragment or variant (such as an analogue, derivative or mutant) is capable of exercising the same or improved physiological function as the heterologous protein. Such variants include naturally occurring allelic variants and non-naturally occurring variants. Unless otherwise specified, additions, deletions, substitutions and derivations of one or more of the amino acids are contemplated so long as the modifications do not result in loss of functional activity of the fragment or variant. Preferably the functionally active peptide or variant has at least about 80% sequence identity more preferably at least about 90% sequence identity, even more preferably at least about 95% sequence identity, most preferably at least about 98% sequence identity to the active and relevant part of the heterologous protein.

[0054] When the immunomodulator refers to a peptide, it refers to a peptide which modifies an immune response by stimulating or suppressing it, for example, activating T-cells, inducinginfiltration of T-cells, recruiting immune cells, etc. In the context of the given description, a heterologous immunomodulator is a therapeutic agent, such as LIGHT, GMCSF, SIRP alpha, or IL 18, which can be used to enhance or suppress the immune system's response to a tumor or disease. The heterologous peptides do not belong to the bacterial proteome but typically belong to a mammal and specifically a human, and are genetically incorporated into the bacterial chromosome or plasmid. Specific immunomodulators in the present disclosure may include IL 18, SIRP alpha, GMCSF and LIGHT.

[0055] IL 18, SIRP alpha, GMCSF and LIGHT may refer to a peptide or polynucleotide encoding the protein or parts thereof, depending on the context. As used herein, the IL 18, SIRP alpha, GMCSF and LIGHT peptides may refer to variants not specifically listed in the present disclosure. Identification of variants which optimize function can be made through by standard screening assays. Alternative fragments of the peptide may exhibit biological activity or even improve interaction time, turnover and / or recycling to optimize activity. In addition, variants may be incorporated into the fusion protein complex of the invention to optimize expression and / or stability of the binding domain. Similarly, the expression constructs could be modified (i.e., codon optimization, removal of secondary structures) to improve gene expression, translation, post translational modification and / or secretion.

[0056] As used herein, aspirin, chemically known as acetylsalicylic acid (herein “ASA”) is primarily recognized as an analgesic, antipyretic, and anti-inflammatory medication. The term is used throughout the disclosure as an inducer for the aspirin-inducible prokaryotic expression cassettes or the immunomodulator fusion peptides, however, it is meant to include induction by chemical modifications to aspirin or alternative salicylic acid or salicylate derivatives as one of ordinary skill in the art would appreciate that these alternatively function as inducers to a similar extent.

[0057] The term “expression” or “secretion” refers to an amount of the at least one heterologous factor with detectable presence inside or outside of the bacterial cells. The term “secretion” refers to an amount of the at least one heterologous factor with detectable presence outside of the bacterial cells.

[0058] The term “biologically active” or “biological activity” of said heterologous immunomodulators refers to the ability to bind a target domain in vitro or activate a target pathway in vitro. Alternatively, it may refer to the ability to change the immune profile of a tumorin vivo or induce increased treatment efficacy in vivo when expressed as a single factor or in combination with other heterologous factors.

[0059] It will be readily understood by those skilled in the art and it is intended here, that when reference is made to particular signal peptides sequence listings, such reference includes sequences which substantially correspond to its complementary sequence and those described including allowances for minor sequencing errors, single base changes, deletions, substitutions and the like, such that any such sequence variation corresponds to the nucleic acid sequence of the signal peptide or other peptide / protein to which the relevant sequence listing relates.

[0060] As used herein, “percent homology”, “percent identity”, "sequence identity", or "identity" or grammatical equivalents as used herein in the context of two nucleic acid or polypeptide sequences includes reference to the residues in the two sequences which are the same when aligned. When percentage of sequence identity is used in reference to proteins it is recognized that residue positions which are not identical often differ by conservative amino acid substitutions, where amino acid residues are substituted for other amino acid residues with similar chemical properties (e.g., charge or hydrophobicity) and therefore do not change the functional properties of the molecule. Where sequences differ in conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. Sequences which differ by such conservative substitutions are considered to have "sequence similarity" or "similarity". Means for making this adjustment are well-known to those of skill in the art. Typically this involves scoring a conservative substitution as a partial rather than a full mismatch, thereby increasing the percentage sequence identity. Thus, for example, where an identical amino acid is given a score of 1 and a non-conservative substitution is given a score of zero, a conservative substitution is given a score between zero and 1. The scoring of conservative substitutions is calculated, e.g., according to the algorithm of Henikoff S and Henikoff JG. (1992).

[0061] Percent identity can be determined using any homology comparison software, including for example, the BlastN software of the National Center of Biotechnology Information (NCBI) such as by using default parameters.

[0062] Other exemplary sequence alignment programs that may be used to determine % homology or identity between two sequences include, but are not limited to, the FASTA package (including rigorous (SSEARCH, LALIGN, GGSEARCH and GLSEARCH) and heuristic (FASTA, FASTX / Y, TFASTX / Y and FASTS / M / F) algorithms, the EMBOSS package (Needle,stretcher, water and matcher), the BLAST programs (including, but not limited to BLASTN, BLASTX, TBLASTX, BLASTP, TBLASTN), and megablast. In some embodiments, the sequence alignment program is BLASTN. For example, 95% homology refers to 95% sequence identity determined by BLASTN, by combining all non-overlapping alignment segments (BLAST HSPs), summing their numbers of identical matches and dividing this sum with the length of the shorter sequence. Other methods are known to the skilled artisan.

[0063] As used herein the term "method" refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts.

[0064] As used herein, the term “treating” includes abrogating, substantially inhibiting, slowing or reversing the progression of a tumor, substantially ameliorating clinical or aesthetical symptoms of a tumor growth, stabilizing disease, extending progression-free survival, extending the overall survival or the extending disease free survival. Particular subjects which are treated are mammalian subjects - e.g., humans. According to a particular embodiment, the subject has been diagnosed as having cancer. Treating cancer in a subject may involve reducing tumor volume, halting tumor progression, alleviating cancer-related symptoms, or extending overall survival or the disease free survival. Alternatively, or additionally, reducing tumor volume may refer to any reduction in cancer metastasis.

[0065] As used herein, administering refers to any systemic administration route known in the art such as delivery of a substance into the bloodstream, typically through oral ingestion, injection. Injection refers to transvenous injection, peritoneal injection, subcutaneous injection, intramuscular injection, or intra-tumoral injection.

[0066] The contents of all cited references and patents are hereby incorporated by reference in their entirety. The invention is explained in more detail by means of the following examples without, however, being restricted thereto.

[0067] It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination, or in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub combination or as suitable in any other described embodiment of the invention. Certain featuresdescribed in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment is inoperative without those elements.

[0068] Various embodiments and aspects of the present invention as delineated herein and as claimed in the claims section below find experimental support in the following examples.

[0069] The term "attenuated" refers to a bacterium rendered to be less virulent compared to the native bacterium, thus becoming harmless or less virulent. The term "attenuated" further refers to a bacterium which is modified to reduce virulence, toxicity, pathogenicity, tumor-homing, and / or antibiotic resistance. Modification may involve a mutation, deletion, reduced expression, or a less active product of a specific gene or set of genes as compared to non-attenuated bacteria. The deletion may refer to providing a nucleic acid molecule capable of inhibiting or deleting the expression of an endogenous gene. Preferably, the ability to home to a tumor is not reduced by the attenuation, such that homing ability is not reduced by more than 80 %, more preferably 70 %, more preferably 60 % more preferably 50 %, more preferably 40 %, more preferably 30 %, more preferably 20 %, more preferably 10 % as compared to non-attenuated (native bacteria) following i.v. administration (e.g., in a mouse model).

[0070] As used herein, the biologically active form refers to the form of a peptide that is capable of exerting a biological effect in a living organism. In the context of the described tumor-homing bacterium, the biologically active form refers to the immunomodulator fusion peptides that are expressed by the inducible prokaryotic expression cassettes. These peptides, when secreted by the bacteria, can exit the bacteria cell and interact with the immune system to modulate its response, potentially aiding in the tumor reduction.

[0071] An immunomodulator fusion peptide refers to a heterologous immunomodulator that is linked to a secretion signal and may form part of a set of immunomodulator fusion peptides. The heterologous immunomodulator may be any number of peptides including LIGHT, GMCSF, SIRP alpha, and IL 18. The fusion peptides are typically designed to enhance the immune response against cancer cells, and their secretion signals facilitate the delivery of these peptides to the tumor environment. The polynucleotide encoding the immunomodulator fusion peptide is further associated with a ribosomal binding site or a cleavage site.

[0072] As used herein, a tumor-homing bacterium or bacteria refers to a population of recombinant bacterium that naturally target and colonize the tumor tissue or that has been engineered to specifically target and colonize tumor tissue.

[0073] Modified to reduce virulence, toxicity, pathogenicity, tumor-homing, and / or antibiotic resistance may involve a mutation, deletion, reduced expression, or a less active product of a specific gene or set of genes as compared to non-attenuated bacteria of the species Salmonella enterica, as disclosed in PCT / IL2023 / 050876. In one example, the modification may be a mutation, deletion, reduced expression, or a less active product of the gene STM3120 (SEQ ID NO 1), also known as STM14_3769 locus in S typhimurium 14028 strain. In another example, the modification may involve a modification in multiple genes selected from the group consisting of STM3120, arginine deiminase (adI)(SEQ ID NO 2), L-asparaginase II (ansB) (SEQ ID NO 3), Aminoglycoside (3") (9) adenylyl transferase (aadA)(SEQ ID NO 4), AAC(6’)-Iaa (aac6) (SEQ ID NO 5)and Tetrathionate reductase A (ttrA) (SEQ ID NO 6). In another example, the modification may involve a mutation, deletion, expression reduction or a less active product of at least three genes selected from the group consisting of STM3120, arginine deiminase (adl), L- asparaginase II (ansB), Aminoglycoside (3") (9) adenylyl transferase (aadA), AAC(6’)-Iaa (aac6) and Tetrathionate reductase A (ttrA) as compared to non-attenuated bacteria of the species Salmonella enterica. In another example, the modification may involve a mutation, deletion, expression reduction or a less active product of at least three genes selected from the group consisting of STM3120, arginine deiminase (adl), ), L-asparaginase II (asnB), Aminoglycoside (3") (9) adenylyl transferase (aadA), AAC(6’)-Iaa (aac6) and Tetrathionate reductase A (ttrA) as compared to non-attenuated bacteria of the species Salmonella enterica. Although Salmonella enterica may be referred to specifically, it is to be recognized that any bacteria having homologous genes can be similarly modified with genomic mutation, deletions, or expression reduction as described herein.

[0074] As used herein, cancer associated antigens may refer to molecules expressed by cancer cells that can be recognized by the immune system. These antigens may be proteins, glycoproteins, or other biomolecules that are preferentially expressed, overexpressed, mutated, or otherwise altered in cancer cells compared to normal cells. Cancer associated antigens may include tumor-specific antigens, tumor-associated antigens, and neoantigens. Cancer associated antigens may be present on the cell surface, secreted, or located intracellularly, and may vary depending on the type and stage of cancer. Tumor-specific antigens may refer to proteins or other molecules that are uniquely expressed by cancer cells and are not found in normal cells. These antigens may arise from mutations or other genetic alterations specific to tumor cells, and may be recognized as foreign by the immune system. Tumor-associated antigens may refer to proteinsor other molecules that are expressed at higher levels in cancer cells compared to normal cells, but are not exclusively found in tumors. These antigens may be overexpressed or abnormally expressed in cancer cells, and while they may also be present in some normal tissues, their elevated expression in tumors may make them potential targets for cancer therapies or immune responses. Examples of cancer associated antigens include for example, Ova, mastl, KRAS G12D, P53 R175H, NRAS Q61K, ADPGK, Ova, KRAS G12D, ADPGK in SEQ ID NOs 12 -20. Examples of neoantigens are known in the art and include, for example, mutant APC antigen as set forth in SEQ ID NO 7 is QATEAERSF. Examples of BRCA mutated epitopes are YIHTHTFYV (SEQ ID NO 8) and SQIWNLNPV (SEQ ID NO 9). An example of a universal HLA-DR-binding T helper synthetic epitope (AKFVAAWTLKAAA) (SEQ ID NO 10) is the pan DR-biding epitope (PADRE), which is a 13 amino acid peptide that activates CD4+ T cells. Another contemplated cancer-associated antigens may include MAGE1 , MAGE-A, survivin, and MUC1 which may be over-expressed in many patients.

[0075] As used herein, a homologous cancer associated antigen is a peptide or polynucleotide encoding the peptide or set of peptides. A polynucleotide may include one or multiple cancer associated antigens of a given tumor or of multiple cancer types. The term homologous cancer associated antigen may refer to a similar but distinct single or series of cancer associated antigen compared with a second single or set of cancer associated antigen. A similar but distinct single nucleotide may refer to codon shuffling without effecting the amino acid sequence. A similar series of neoantigens may involve a polynucleotide encoding a series of reordered neoantigens. For example, if a prokaryotic expression cassette includes a series of three neoantigen encoded in the order of A-B-C, then a homologous series of the series of neoantigens may include the same three neoantigens in a different order, such as B-A-C. In some embodiments, the homologous series of neoantigens may involve both codon shuffling as well as reordered neoantigens. In some embodiments, when a number of different homologous neoantigen polynucleotides are chromosomally integrated, each homologous neoantigen polynucleotide may be distinct but homologous to the other. The series of neoantigens may not include a cleavage site nor linker between each respective neoantigen. In some embodiments, neoantigen or set thereof, and each homologous neoantigen or set thereof, are typically operably linked to a constitutive prokaryotic promoter.

[0076] As used herein, a neoantigen can refer to a polypeptide sequence or a nucleotide sequence. In the context of a peptide, the neoantigen is an epitope that has at least one alterationthat makes it distinct from the corresponding wild-type antigen. The alteration may be derived from a mutation in a tumor cell or a post-translational modification specific to a tumor cell. The mutation may have caused a loss or a gain of function of its original cellular function. A mutation can include a frameshift or non-frameshift deletion, missense or nonsense substitution, splice site alteration, genomic rearrangement or gene fusion, or any genomic or expression alteration giving rise to a neoORF. Examples of neoantigens are known in the art and include, for example, mutant APC antigen as set forth in SEQ ID NO 7 is QATEAERSF. Examples of BRCA mutated epitopes are YIHTHTFYV (SEQ ID NO 8) and SQIWNLNPV (SEQ ID NO 9). An example of a universal HLA-DR-binding T helper synthetic epitope (AKFVAAWTLKAAA) (SEQ ID NO 10) is the pan DR-biding epitope (PADRE), which is a 13 amino acid peptide that activates CD4+ T cells. Another contemplated cancer-associated neoantigen is the GL261 neoantigen (mlmp3 D81N, sequence AALLNKLYA) (SEQ ID NO 11).

[0077] A neoantigen polynucleotide may include a single or multiple neoantigens of a given tumor or of multiple cancer types.

[0078] As used herein, homologous neoantigen polynucleotide refers to a similar but distinct single neoantigen or set of neoantigen compared with another single or set of neoantigen polynucleotide. A similar but distinct single nucleotide may refer to codon shuffling without effecting the amino acid sequence. A similar series of neoantigens may involve a polynucleotide encoding a series of reordered neoantigens. For example, if a prokaryotic expression cassette includes a series of three neoantigen encoded in the order of A-B-C, then a homologous series of the series of neoantigens may include the same three neoantigens in a different order, such as B- A-C. In some embodiments, the homologous series of neoantigens may involve both codon shuffling as well as reordered neoantigens. In some embodiments, when a number of different homologous neoantigen polynucleotides are chromosomally integrated, each homologous neoantigen polynucleotide may be distinct but homologous to the other. The series of neoantigens may lack a cleavage site or linker between each respective neoantigen. In some embodiments, a neoantigen and homologous neoantigen, are each operably linked to a constitutive prokaryotic promoter.

[0079] As used herein, the one or more prokaryotic expression cassettes, constitutive prokaryotic expression cassettes or inducible prokaryotic expression cassettes includes two or more polynucleotides. This language is meant to include multiple scenarios wherein the two ormore polynucleotides are incorporated into a single prokaryotic expression cassette or multiple prokaryotic expression cassettes.

[0080] As used herein, a constitutive prokaryotic promoter is active in a cell in all circumstances, while an inducible promoter becomes active in response to a specific inducer, such as aspirin. A promoter is a nucleic acid sequence that initiates transcription of a particular gene. Promoters are operably linked to a polynucleotide sequence and are typically located near the transcription start side of the gene, on the same strand and upstream of the DNA, which is towards the 5' region of the same strand which is to be transcribed. The constitutive prokaryotic promoter may be naturally present in the bacteria and operably linked during the engineering process. Alternatively, it may be introduced as part of the prokaryotic expression cassette introduced into the bacterium.

[0081] Preferably, the respective promoter used is an autologous promoter from a tumorhoming bacterium, which expresses the respective nucleic acid sequence. Alternatively, the respective promoter is a heterologous promoter, preferably a prokaryotic promoter.

[0082] As used herein, a prokaryotic expression cassette may be organized to include a promoter, a secretion or transport signal, protein or peptide or alternatively a promoter, protein / peptide, secretion or transport signal. A prokaryotic expression cassette may further include a cleavage site between the peptide and secretion signal or transport signal which may allow its separation from the protein or peptide upon secretion, thereby minimizing spatial interference and maximizing performance.

[0083] The terms “transport signal”, “signal peptide" and "signal sequence" may be used interchangeably herein and unless otherwise indicated, refers to an amino acid sequence or the nucleotide sequence encoding the amino acid sequence which direct the localization of a peptide such that it is secreted from, or displayed on the surface of the outer membrane of the bacterium (,i.e., surface display). The transport signal used herein may facilitate secretion of the protein from the cell in which it is produced. The sequence is associated with or linked to a peptide set forth herein or the nucleotide sequence encoding the peptide sequence. The sequence may be associated with or linked at C or N-terminus. The proteins or peptides may be a single neoantigen, a set of neoantigens or a concatemer of neoantigens. The proteins or peptides may alternatively be a single heterologous immunomodulator.

[0084] As used herein, “secretion” of a protein refers to the transportation of a heterologous peptide outward across the double membrane of a recombinant Gram-negative bacterium or Gram-negative bacterial strain.

[0085] For illustration purposes, referring to FIG. 4, 310 is the intracellular space of a bacteria, while 311 is the periplasm and 312 is outside the bacterial call.

[0086] Double membrane-spanning secretion systems which span the inner and outer membranes as depicted in FIG. 4, 303 and include, but are not limited to, a type I secretion system (T1SS), a type III secretion system (T3SS), a type IV secretion system (T4SS), a type VI secretion system (T6SS), and a resistance-nodulation-division (RND) family of multi-drug efflux pumps (Costa et al., 2015), incorporated herein by reference. Double membrane-spanning secretion systems generally transport peptides from the bacterial cytoplasm directly into the extracellular space or into the target cell.

[0087] In contrast, some secretion systems, such as Type II secretion system, T2SS (see FIG. 4, 302 with 301), Type V secretion system - T5SS, Type VIII secretion system - T8SS, and Type IX - T9SS employ a two-step pathway to first transfer proteins through the inner membrane, using the Sec system or the Tat secretion system, which spans the inner membrane such that peptides are translocated into the periplasm by inner membrane-spanning transporters. Subsequently, they may transport via the outer membrane.

[0088] Inner membrane secretion systems, illustrated in FIG. 3A, 302, refer to translocation through inner membrane-spanning transporters and into the periplasm of a given bacterium. In the present disclosure, an "inner membrane secretion signal" may refer to a signal sequence that directs the secretion of a heterologous immunomodulator peptides to the inner membrane of the tumor-homing bacterium, facilitating its release and potentially its interaction with the host's immune system. Examples include sec and tat secretion signals which employ the sec or tat secretion systems.

[0089] Double membrane secretion signals refers to a specialized signaling mechanism that directs the secretion of heterologous immunomodulators across two membranes of a given Gramnegative bacterium. This system may involve a single-step or two-step secretion machinery. In the two step process, the immunomodulators are first targeted to one membrane and then transported to another, for delivery of these therapeutic agents to the tumor site. Outer membranespanning secretion systems include, but are not limited to, a type V autotransporter secretionsystem (T5SS), a curli secretion system, and a chaperone-usher pathway for pili assembly (Costa et al., 2015), as depicted in FIG. 4, 304. Examples include the Type V secretion system.

[0090] In the single-step process, the immunomodulators are transported through both membranes directly as illustrated in FIG. 4, 303. Examples include the Type I secretion system, Type III secretion system, and the Type IV secretion system.

[0091] A Gram-negative bacterium or Gram-negative bacterial strains used herein usually comprise bacterial proteins constituting essential components of the inner membrane secretion system (e.g., sec or tat system of the bacterial type II secretion system (T2SS)) as well as the double membrane secretion system such as type III secretion system (T3SS), and / or the type V secretion system (T5SS). Unless otherwise indicated, a transport signal may belong to any inner or double membrane secretion system including for example, Sec or tat secretion system of the Type II secretion system, Type III secretion system, Type V secretion system and outer cell wall display system of a negative gram bacterium. For example, the sec or tat secretion system refers to a protein secretion system composed of two proteins: a membrane- spanning complex called the Sec or Tat translocase, and a sec / tat secretion signal also referred to as the Signal Recognition Particle (SRP). The Sec system facilitates the co-translational and post-translational import of unfolded proteins into the periplasmic space, while the Tat system is responsible for the transport of fully folded proteins across the inner membrane and into the periplasmic space.

[0092] As used herein, a sec secretion signal may refer to a specific amino acid sequence at the N-terminus of a protein that directs the protein to the Sec translocase for transport across the bacterial inner membrane. The Sec pathway typically transports unfolded proteins. A tat secretion signal may refer to a specific amino acid sequence at the N-terminus of a protein that directs the protein to the twin-arginine translocation (Tat) system for transport across the bacterial inner membrane. The Tat pathway typically transports folded proteins and is characterized by a conserved twin-arginine motif in the signal peptide.

[0093] It will be readily understood by those skilled in the art and it is intended here, that when reference is made to a particular transport signal sequence listing, such reference includes sequences which substantially correspond to its complementary sequence and those described, including allowances for minor sequencing errors, single base changes, deletions, substitutions and the like, such that any such sequence variation corresponds to the nucleic acid sequence of the signal peptide or other peptide / protein to which the relevant sequence listing relates.

[0094] Transport signals belong to different bacterial transport systems which are made up of bacterial effector proteins. Transport signals direct secretion or display on the bacterial surface, also referred to as a surface display signal. Transport signals typically direct localization of a protein such that it is secreted from, or positioned on, the outer membrane of the bacteria. Transport signals typically direct localization of a protein by way of an mRNA structure or protein sequence motif, as is the case for the MISSSSIS sequence (SEQ ID NO 14) or alternatively, a leader sequence on the C- or N-terminus which may be cleaved. For example, a transport signal may facilitate surface display of the peptide on the outer wall of the bacteria by directing localization of a peptide to the exterior surface of the outer membrane. Alternatively, depending on the transport signal, a given transport signal may be a secretion signal which facilitates any one of a number of stages in the secretion of a peptide from a bacterial cell in which it is produced. Typically, the bacterial host cell engineered to express the fusion peptide or protein linked to the secretion signal is a bacterium having a functional secretion system of that signal. Transport signals may be cleaved from the remainder of the protein, often referred to as the mature protein, upon secretion from the cell however, in some cases, there is no protease cleavage site between the transport signal and the neoantigen or heterologous immunomodulator.

[0095] When the transport signal is a surface display signal, the bacteria display the peptide (e.g., neoantigen) on the bacterial surface using a bacterial surface display system. Examples of bacterial surface display systems include outer membrane protein systems (e.g., LamB, FhuA, Ompl, OmpA, OmpC, OmpT, eCPX derived from OmpX, OprF, and PgsA), surface appendage systems (e.g., F pillin, FimH, FimA, FliC, and FliD), lipoprotein systems (e.g., INP, Lpp-OmpA, PAL, Tat- dependent, and TraT), and virulence factor-based systems (e.g., AIDA-1, EaeA, EstA, EspP, MSP1 a, and invasin). Exemplary surface display systems are described, for example, in van Bloois, E., et al., Trends in Biotechnology, 2011 , 29:79-86, which is hereby incorporated by reference. For example, the bacteria may be engineered to display the RGD peptide sequence (ACDCRGDCFCG SEQ ID NO 13) on the external loop of outer membrane protein A (OmpA).

[0096] Secretion signals may be categorized as belonging to a transport system selected from: a surface display system, an inner membrane secretion system, a double membrane secretion system which include a specialized protein system, which directs the export of a protein, or alternatively, a wall presenting system. For example, the sec or tat secretion system transports a given peptide through the inner lipid membrane but not through the outer lipid membrane. In this case, the peptide is deposited in the periplasmic space between the inner and outer membranes.

[0097] Secretion signals of bacterial Type III secretion systems are known by those skilled in the art and may include Ssphl (SEQ ID NO 61), Ssph2, MISSSSIS (SEQ ID NO 14), sigE, sigE / pipC (SEQ ID NO 67), sopB (SEQ ID NO 68), sopA (SEQ ID NO 66), invB (SEQ ID NO 65), SptP (SEQ ID NO 64), SipA (SEQ ID NO 69), sicP (SEQ ID NO 63), SipB (SEQ ID NO 70), SipC, SipD, InvJ, SpaO, AvrA, and SopE secretion signals of Salmonella, the YopE, YopH, YopM and YpkA secretion signals of Yersinia spp., the Ipa secretion signals of Shigella, and the ExoS secretion signals of Pseudomonas aeruginosa. In some embodiments, the secretion signals of bacterial type III secretion systems are selected from MISSSSIS, sspH2, sspHl, sigE-sopB and sipB.

[0098] Secretion signals of the bacterial Type II secretion system (secreted out of the bacteria) e.g., PelB DNA sequence (SEQ ID NO 15), are known by those skilled in the art. Some examples include pelB (SEQ ID NO 62), ompA, PSP (SEC family), yebF.

[0099] Secretion signals of bacterial Type V secretion systems are known by those skilled in the art. They include for example, the PET autotransporter. Type V secretion signals may utilize an N-terminal Sec-dependent secretion signal which facilitates transport across the inner membrane and a C-terminal tag which facilitates transport across the outer-membrane. This system uses the Sec-system to get from the cytoplasm to the periplasm. The C-terminal tag then inserts into the outer membrane forming a pore of the beta-barrel structure ahead of the linker sequence through which the “passenger protein” threads through. Once across the outer membrane, the neoantigen or series thereof may be released from the membrane-embedded C- terminal tag by either an autocatalytic, or a membrane-bound protease.

[0100] A cleavage site refers to a site within a protein or peptide sequence where a specific enzymatic reaction, such as proteolysis, occurs. In the context of the immunomodulator fusion peptides, the cleavage site typically refers to a sequence that is recognized and cleaved by a specific protease, allowing for the release of the active peptide.

[0101] A constitutive prokaryotic expression cassette refers to a sequence of genetic elements used to facilitate the constitutive expression of a peptide of interest in prokaryotic cells. The sequence of genetic elements will include a constitutive prokaryotic promoter that is constantly active, without dependence on an inducer, and initiates transcription of the polynucleotide in a prokaryotic organism.

[0102] A deactivating mutation refers to a mutation that results in the loss of function of the gene product. In the context of the provided disclosure, a deactivating mutation would refer tochanges in the gene sequence that leads to the production of a nonfunctional outer membrane protein or a compromised of the outer membrane integrity. For example, a deactivating mutation of an outer membrane protein may results in a leaky or destabilized outer membrane. This feature has been claimed by others to be critical for the facilitation of translocation of therapeutic polypeptides from the periplasm to the extracellular space when an inner membrane secretion system is employed or when an inner membrane secretion signal is fused to a peptide. Deactivating mutations are known in the art and may include one or more genes encoding a protein that tethers the outer membrane to the peptidoglycan skeleton, such as ompA, ompF, tolA, tolB, pal.

[0103] As used herein, the term "trans position" of the regulating prokaryotic expression cassette may refer to a configuration where the regulating expression cassette encoding a regulator is located at a different genetic locus or on a separate genetic element (e.g., a plasmid) from the one or more inducible prokaryotic expression cassettes it regulates. In this arrangement, the regulator may act on the inducible promoters from a distance, allowing for independent control and modulation of gene expression.

[0104] As used herein, an "effective amount" may refer to a quantity of bacteria that may be sufficient to produce a desired therapeutic effect while minimizing potential adverse effects (i.e., having a reasonable benefit / risk ratio). The effective amount may vary depending on factors such as the specific therapeutic goal, the subject's age and health status, treatment duration, concurrent therapies, and the particular bacterial strain used. In some cases, the effective amount may be the minimum quantity needed to achieve the intended outcome. The effective amount may range from about 10^5 to 10^8 bacteria per square meter of body surface area in some implementations.

[0105] As used herein, an immune checkpoint modulator is a class of drugs that inhibit immune checkpoints, which are regulators of activated T cells. The immune checkpoint modulator completely or partially reduce, inhibit, interfere with, or negatively affect the function of one or more checkpoint molecules. These drugs are used to treat cancer by blocking certain proteins made by some immune cells, such as T cells, and some cancer cells, allowing an immune response against the cancer cells. Examples include PD-1 / PD-L1 inhibitors and CTLA-4 inhibitors.DETAILED DESCRIPTION

[0106] The invention is based, at least in part, on the discovery that tumor-homing bacterium can be genetically modified for aspirin induced secretion of a set of heterologousimmunomodulators. Despite challenges relating to tumor-homing and bacterial secretion of heterologous immunomodulator at the tumor site, a single induction by aspirin in a subject upon tumor-homing or a reduction in a circulatory bacterial load was sufficient to have a significant effect on tumor growth in a subject. Specifically, the anti-inflammatory effects typically exhibited by aspirin, do not inhibiting the tumor-local immunomodulation of a set of immunomodulators secreted by the tumor-homing bacterium.

[0107] Embodiments consistent with the present disclosure provide vaccines, genetically modified tumor-homing bacterium, and methods of treatment using a tumor-homing bacterium genetically modified for exogenously induced, tumor-locally delivered, and timely-controlled delivery of a set of heterologous immunomodulator.

[0108] In a first aspect, there is provided a tumor-homing bacterium, and preferably a Gram negative bacterium, genetically modified to include a regulating expression cassette as well as one or more inducible prokaryotic expression cassettes. The one or more inducible prokaryotic expression cassettes may include: a directly or indirectly inducible promoter that is not associated with the prokaryotic expression cassette in nature, and associated with an immunomodulator expression cassette, said immunomodulator expression cassette comprising a polynucleotide encoding a single or series of heterologous immunomodulators, wherein the inducible promoter is induced by a small molecule that is safe to administer to a subject, and wherein each polynucleotide encoding a heterologous immunomodulator is associated with a ribosomal binding site (supporting a polycistronic mRNA conformation) or cleavage site and optionally associated with a polynucleotide encoding a transport signal from a transport system. In some embodiments, the regulating expression cassette may include a heterologous exogenously inducible transcription regulator selected from the list consisting of: araC (arabinose), Lad (IPTG), salR or nahR (acetylsalicylic acid (ASA)).

[0109] In another aspect, there is provided a tumor-homing bacterium, genetically modified for inducible expression of a heterologous immunomodulator including a regulating expression cassette encoding a regulator; and one or more inducible prokaryotic expression cassettes comprising: a directly or indirectly inducible promoter associated with an immunomodulator expression cassette having a polynucleotide encoding a single or series of heterologous immunomodulators selected from a list consisting of: LIGHT, GMCSF, SIRP alpha, and IL18, wherein the inducible promoter is induced by a small molecule that is safe to administer to a subject. In some embodiments, the inducible promoter is induced by a small molecule selectedfrom the list consisting of L-arabinose, IPTG, or a salicylic acid, acetylsalicylic acid or derivative thereof. In some embodiments, the inducer is regulated by a salicylic acid, acetylsalicylic acid or derivative thereof. In some embodiments, the inducible promoter is induced by a small molecule that is safe for humans, including but not limited to, araC-pBAD (induced by L-arabinose), LacI-pLac (induced by IPTG), SalR-pSal or nahR-pSal (induced by acetylsalicylic acid (ASA)). In some embodiments, the small molecule is a salicylate, salicylic acid or derivative thereof (e.g., acetylsalicylic acid). In some embodiments, the inducer is regulated by a salicylic acid, acetylsalicylic acid or derivative thereof.

[0110] Embodiments consistent with the present disclosure provide vaccines, genetically modified tumor-homing bacterium, and methods of treatment using tumor-homing bacterium genetically modified for aspirin inducible tumor-local delivery of multiple heterologous immunomodulators. The tumor-local and dose efficacious delivery of biologically active heterologous immunomodulators provide for more limited systemic side effects. Embodiments consistent with the present disclosure provide a tumor-homing bacterium genetically modified to include multiple prokaryotic expression cassette. In some embodiments, the multiple prokaryotic expression cassette are present on a plasmid or chromosomally integrated. In some embodiments, the multiple expression cassettes may be chromosomally integrated prokaryotic expression cassette. According to an aspect of the present disclosure, a tumor-homing bacterium is provided. The tumor-homing bacterium is genetically modified to include a total of three or more prokaryotic expression cassettes: i. a constitutive prokaryotic expression cassette encoding two homologous cancer associated antigens or specifically homologous neoantigens, each associated with a transport signal from a distinct transport system, ii. a regulating expression cassette encoding a regulator; and iii. an inducible prokaryotic expression cassette comprising an inducible transcription promoter and a polynucleotide encoding a single or series of immunomodulator peptides selected from a list consisting of: LIGHT, GMCSF, SIRP alpha, and IL 18, and wherein each polynucleotide encoding an immunomodulator peptide is associated with a polynucleotide encoding a ribosomal binding site.

[0111] According to other aspects of the present disclosure, the tumor-homing bacterium may include one or more of the following features. The inducible prokaryotic expression cassette may comprise a polynucleotide encoding a LIGHT peptide, said LIGHT peptide being a portion of an extracellular domain of LIGHT. The inducible transcription promoter and regulator may be induced by a small molecule that is safe for humans, including but not limited to, L-arabinose,IPTG, or salicylic acid or derivative thereof. The small molecule may be regulated and induced by a salicylic acid, acetylsalicylic acid or derivative thereof. Upon induction, a light peptide may be secreted or expressed in a biologically active form. The regulating expression cassette encoding a regulator may be in trans position relative to the inducible prokaryotic expression cassette. The polynucleotide encoding a light peptide may be associated with a transport signal from a transport system selected from a group consisting of: Type II secretion system, Type III secretion system, Type V secretion system and outer cell wall display system of a negative gram bacteria. The polynucleotide encoding the polynucleotide encoding a LIGHT peptide may be associated with a transport signal from a Type II secretion system. A 2: 1 ratio or more may exist between a number of inducible prokaryotic expression cassettes and a number of regulating expression cassette.

[0112] The inducible prokaryotic expression cassette(s) may further comprise a polynucleotide encoding an IL 18 peptide. Two different inducible prokaryotic expression cassettes may comprise the polynucleotide encoding an IL 18 peptide and the polynucleotide encoding a LIGHT peptide. A single inducible prokaryotic expression cassette may include a polynucleotide encoding an IL18 peptide and a LIGHT peptide. A polynucleotide encoding a IL18 peptide may be associated with a polynucleotide encoding a secretion signal from a Type III, Type II or Type V secretion system. In preferred embodiments, the polynucleotide encoding the IL 18 peptide may be associated with a polynucleotide encoding a secretion signal from a Type III secretion system. The inducible prokaryotic expression cassette(s) may further comprise a polynucleotide encoding a GMCSF peptide. Two different inducible prokaryotic expression cassettes may comprise a polynucleotide encoding a IL 18 peptide, a polynucleotide encoding a light peptide and a polynucleotide encoding the GMCSF peptide. A single inducible prokaryotic expression cassette may include a polynucleotide encoding an IL 18 peptide, a LIGHT peptide and a GMCSF peptide. The GMCSF peptide may be associated with secretion signal from a Type II secretion system.

[0113] The inducible prokaryotic expression cassette(s) may further comprise a polynucleotide encoding a SIRP alpha peptide. Two or more inducible prokaryotic expression cassettes may comprise the polynucleotide encoding a IL18 peptide, the polynucleotide encoding a light peptide, the polynucleotide encoding a GMCSF peptide and the polynucleotide encoding the SIRP alpha peptide. A single inducible prokaryotic expression cassette may include a polynucleotide encoding an IL 18 peptide, a LIGHT peptide and a SIRP alpha peptide. The SIRPalpha peptide may be associated with secretion signal from a Type II secretion system. The inducible prokaryotic expression cassette may be inserted at an adl, ttrA or Stm3120 locus or a homology thereof. The immunomodulator expression cassette may be inserted the adl locus or homology thereof. The tumor-homing bacterium may be adapted to provide induced expression and / or secretion of a peptide after 18 days. The transport signal may be from a Type III and Type I secretion system. The multiple expression cassettes may be chromosomally integrated. The bacterium may be selected from a genus being Salmonella and / or Pseudomonas. The bacterium may be selected from a species being Salmonella enterica is modified to include a deletion (null mutation) in Stm3120 or a homology thereof. The bacterium may be a Salmonella Typhimurium.

[0114] According to another aspect of the present disclosure, a tumor-homing bacterium is provided. The tumor-homing bacterium is genetically modified to include a total of three or more prokaryotic expression cassettes: i. a constitutive prokaryotic expression cassette encoding two homologous cancer associated antigens or specifically two homologous neoantigens, each associated with a transport signal from a distinct transport system, ii. a regulating expression cassette encoding a regulator; and iii. an inducible prokaryotic expression cassette comprising an inducible transcription promoter and a polynucleotide encoding a series comprising: LIGHT, GMCSF, SIRP alpha, and IL 18, and wherein each polynucleotide encoding an immunomodulator peptide is associated with a polynucleotide encoding a ribosomal binding site.

[0115] According to other aspects of the present disclosure, the tumor-homing bacterium may be genetically modified to include a constitutive prokaryotic expression cassette encoding two homologous cancer antigen, each associated with a transport signal from a distinct transport system selected from a Type III and a Type V secretion system. Further details are detailed in P.C.T Patent Application No. PCT / IB2024 / 058038, filed Aug. 21, 2024, the contents of which are incorporated herein by reference in its entirety.

[0116] According to another aspect of the present disclosure, a vaccine is provided. The vaccine comprises a tumor-homing bacterium of any one of the aspects described above and an excipient or carrier.

[0117] According to another aspect of the present disclosure, a method of treating a cancer of a subject in need thereof is provided. The method comprises administering to the subject an effective amount of the vaccine described above.

[0118] According to other aspects of the present disclosure, the method may include one or more of the following features. Treating a cancer may comprise inducing an antigen specificimmune response against a cancer associated antigen (e.g., neoantigen) when administered to a subject in need thereof. Treating a cancer may comprise reducing a tumor volume. The method may further comprise administering a salicylate or salicylic acid. Administering to the subject may be by parenteral administration. The cancer may be selected from a group consisting of breast, melanoma, colorectal cancer, lung cancer, gastric cancer, pancreatic cancer, ovarian cancer, bone cancer and brain cancer.

[0119] In another aspect, there is provided, a tumor-homing bacterium, genetically modified for inducible tumor-local delivery of multiple heterologous immunomodulators. The tumor-homing bacterium is typically an attenuated Gram-negative bacterium.

[0120] In some embodiments, the tumor-homing bacterium is a recombinant bacteria. In some embodiments, The tumor-homing bacterium is further modified to include a directly or indirectly exogenously inducible prokaryotic expression cassette. In preferred embodiments, the exogenously inducible prokaryotic expression cassette is aspirin inducible. In some embodiments, inducible prokaryotic expression cassette encodes one or more immunomodulator fusion peptides, and preferably a set of immunomodulator fusion peptides. Each of said immunomodulator fusion peptides includes a heterologous immunomodulator (i.e., a polynucleotide encoding a heterologous immunomodulator) associated with a secretion signal.

[0121] In some embodiments, the heterologous immunomodulator is selected from a list consisting of: LIGHT, GMCSF, SIRP alpha, and IL18. In some embodiments, the set of heterologous immunomodulators is two or more heterologous immunomodulators selected from a list consisting of: LIGHT, GMCSF, SIRP alpha, and IL 18. In some embodiments, the set of heterologous immunomodulators is three or more heterologous immunomodulators selected from a list consisting of: LIGHT, GMCSF, SIRP alpha, and IL 18. In some embodiments, the set of heterologous immunomodulators includes LIGHT, GMCSF, SIRP alpha, and IL 18.

[0122] In preferred embodiments, the recombinant tumor-homing bacterium is further genetically modified to include one or more constitutive prokaryotic expression cassettes comprising two or more homologous cancer associated antigen (e.g., homologous neoantigen polynucleotides), each associated with a transport signal from a distinct transport system.

[0123] In a particular embodiment, the tumor-homing bacterium is a recombinant bacteria which has been genetically modified to include a total of three or more prokaryotic expression cassette comprising: i. one or more constitutive prokaryotic expression cassettes comprising two or more homologous neoantigen polynucleotides, each associated with a transport signal from adistinct transport system, ii. a regulating expression cassette encoding a regulator; and iii. one or more aspirin-inducible prokaryotic expression cassettes encoding a set of immunomodulator fusion peptides, each of said immunomodulator fusion peptides comprising a heterologous immunomodulator associated with a secretion signal, said heterologous immunomodulator selected from a list consisting of: LIGHT, GMCSF, SIRP alpha, and IL 18.

[0124] Embodiments consistent with the present disclosure provide bacteria adapted by genetic modification, for delivery to a tumor of functionally active heterologous immunomodulators. In some embodiments, the bacteria are genetically modified to provide inducible expression or secretion of biologically active heterologous immunomodulator. Embodiments consistent with the present disclosure provide, a tumor-homing bacterium adapted, upon induction (i.e., administration of an inducer to a host), to secrete and / or express immunomodultor / s in biologically active form. In some embodiments, upon induction, a set of heterologous immunomodulators are secreted and / or expressed in a biologically active form. In some embodiments, the set of heterologous immunomodulators are secreted and / or expressed in a biologically active form upon at least partial tumor colonization in a subject and systemic exposure to a safe dose of a salicylic acid (e.g., acetylsalicylic acid). In some embodiments, induction of the recombinant bacterium by a single and safe dose of aspirin administered to the host, provides an anti-tumor effective dose of a set of heterologous immunomodulators. In preferred embodiments, a set of two or three or four heterologous immunomodulators are selected from a list consisting of: LIGHT, GMCSF, SIRP alpha, and IL 18 are encoded by one or more immunomodulator expression cassettes.

[0125] In certain embodiments, the bacteria are genetically modified for inducible heterologous immunomodulator, such that upon exposure to a safe dose of salicylate, acetylsalicylic acid or another inducer which is administered at least after partial tumor colonization, at least a partial therapeutic effect is observed. For example, a change in immune profile of a tumor may be consistent with a therapeutic effect. Any one of a number of methods may be used which are recognized in the art as indicative of a therapeutic effect, including but not limited to: recruitment of immune cells, increased Ml / M2 ratio, release of immune suppression on macrophage to induce macrophage killing of tumor cells or T-cell infiltration, reduce myeloid derived suppressor cells and, production of antibodies against tumor neoantigens.

[0126] Embodiments consistent with the present disclosure provide an inducible promoter which is pSal. In some embodiments, the inducible promoter has at least 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with PSal SEQ ID NO. 16.

[0127] Embodiments consistent with the present disclosure provide a regulating expression cassette which is SalR. In some embodiments, the genetically engineered bacteria includes one or more gene sequence(s) having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SalR SEQ ID NO 17.

[0128] In some embodiments, the tumor-homing bacterium is adapted to provide a therapeutic effect on a tumor upon at least partial tumor colonization in a subject and administration of a safe dose of a salicylic acid or derivative thereof.

[0129] In some embodiments, each respective polynucleotide encoding a heterologous immunomodulator is associated with a polynucleotide encoding a transport signal from a transport system.

[0130] In one embodiment, a single regulating expression cassette is present. The term “single” in the context of the single regulating expression cassette refers only to the regulation of the immunomodultor peptides. It is understood that there may be additional regulating expression cassettes which regulate other components. The single regulating expression cassette is based on the discovery, for example in the case of aspirin induction, that despite multiple inducible DNA constructs, all encoding peptides or proteins which are non-native to the bacteria and being chromosomally integrated, the aspirin induction is effective in leading to expression and translation of multiple peptides or proteins. In some embodiments, a 2: 1 ratio or more exists between the number of inducible prokaryotic expression cassettes and the regulating expression cassette. In addition, for aspirin induction, a trans position relative to the inducible prokaryotic expression cassettes is surprisingly more effective than a more expected cis position. Thus, in some embodiments, the regulating expression cassette is in a trans position relative to the inducible expression cassettes.

[0131] In an alternative embodiment, a duplicate regulating expression cassette is present.

[0132] The inducible prokaryotic expression cassette or DNA construct includes a heterologous regulator-dependent prokaryotic promoter associated with an immunomodulator expression cassette. The regulator-dependent prokaryotic promoter refers to a regulatory region or polynucleotide that is operably linked, in this case, to the immunomodulator expression cassette, wherein expression of the cassette is induced in the presence of the inducer. In someembodiments, the immunomodulator expression cassette encodes a single heterologous immunomodulator. In this case, the single heterologous immunomodulator is associated with a transport signal and a leading ribosomal binding site. In another embodiment, the immunomodulator expression cassette encodes a series of heterologous immunomodulator, wherein each respective heterologous immunomodulator is associated with a transport signal and a leading ribosomal binding site.

[0133] In some embodiment, the biologically active peptide is actively secreted, for example by way of a secretion signal from a secretion system. In other embodiments, the biologically active peptide is passively secreted.

[0134] In various embodiments, each polynucleotide encoding a heterologous immunomodulator is associated with a polynucleotide encoding a secretion signal. Relevant secretion signals for the various heterologous immunomodulators may include an inner membrane secretion signal (e.g., sec or tat secretion signal) such as OmpA or PelB, and a Type V secretion signal. In some embodiments, the secretion signal associated with the heterologous immunomodulator and especially a heterologous immunomodulator having a significant cysteine content (e.g., more than 1%, 1-4%) is an inner membrane secretion signal such as a sec or tat secretion signal (e.g., OmpA or PelB). In some embodiments, the heterologous immunomodulator may be between 30 and 400 amino acids. In some embodiments, the heterologous immunomodulator may have a hydrophobicity range of between 0.5 to 0.01 on the Kyte-Doolittle hydrophobicity index.

[0135] General examples of transport signals include: sec, PelB signal peptide, DsbA signal peptide, STII signal peptide, OmpA signal peptide, PhoA signal peptide, LamB signal peptide, SpA signal peptide, and PET autotransporter system.

[0136] In some embodiments, the regulator is a positive regulator. Being effective within bacteria, the transcription regulator refers to a prokaryotic transcription regulator. Exogenously inducible is meant to refer to a setting or circumstance in which the exogenously inducible promoter is active to induce expression. The setting or circumstance is the administration of a chemical inducer.

[0137] In some embodiments, the inducible transcription regulator is a directly induced transcription regulator. In some embodiments, the inducible transcription regulator is an indirectly induced transcription regulator.

[0138] Embodiments consistent with the present disclosure provide for Gram-negative bacterium or Gram-negative bacterium. Examples of Gram-negative bacterium include but are not limited to: Salmonella spp., Yersinia spp., Bordetella spp., Escherichia coli, Shigella spp., Burkholderia mallei, Burkholderia pseudomallei and Pseudomonas aeruginosa, Citrobacter, Klebsiella, Neisseria and Pseudomonas. In various embodiments of the present invention, the attenuated bacteria may be a Salmonella or a Pseudomonas strain. The bacterium may be selected from a species being Salmonella enterica and modified to include a deletion or null mutation in STM3 120 or a homology thereof. In various embodiments of the present invention, the attenuated bacteria may be of the serotype Typhimurium (e.g., strain STM3120 (i.e. having a deletion in STM3120).

[0139] As used herein, an attenuated bacterium refers to a bacterium considered pathogenic before attenuation. Examples include, but are not limited to, Salmonella spp., Yersinia spp., Bordetella spp., Escherichia coli, Shigella spp., Burkholderia mallei, Burkholderia pseudomallei and Pseudomonas aeruginosa. In certain embodiments, the attenuated bacteria are Salmonella enterica. In some embodiments, the attenuated bacteria is Salmonella Typhimurium - e.g., the Salmonella Typhimurium attenuated strain VNP20009, Salmonella Typhimurium 14028 strain STM3120 (also referred to herein as STM3120 strain; see for example Arrach et al., (2010), Salmonella Typhimurium 14028 strain STM1414, Pseudomonas aeruginosa (strain CHA-OST) and / or Bacillus Subtillis (strain PY79). In some embodiments, the Salmonella typhimurium is Salmonella typhimurium VPN20009. In some embodiments, the Salmonella typhimurium VPN20009 is a Salmonella typhimurium VPN20009, having a deletion in STM3120. In some embodiments, the Salmonella Typhimurium has no mutation or reduction in expression conferring purine -auxotrophy (e.g., mutation affecting the pur I gene)

[0140] Embodiments consistent with the present disclosure provide a Gram-negative bacterium or Gram-negative bacterium genetically modified by chromosomal integration of two or more, three or more, four or more prokaryotic expression cassette. In some embodiment, two or more prokaryotic expression cassette are chromosomally integrated. In another embodiment, three or more prokaryotic expression cassette are chromosomally integrated. In another embodiment, four or more prokaryotic expression cassette are chromosomally integrated. In addition, despite the multiple insertions of polynucleotides encoding heterologous or Eukaryotic peptides, there was minimal effect on fitness in vitro or minimal negative impact on the viability of the bacteria used before administration to a subject.

[0141] As used herein, prokaryotic expression cassette are configured to be expressed within a prokaryotic cell and secreted outwards. In some embodiments, the bacteria is modified such that a native or heterologous prokaryotic promoter is associated with each of the respective prokaryotic expression cassette. In various embodiments, the prokaryotic promoter is constitutively active. For example, the bacteria may be modified such that a cancer associated antigen or neoantigen is constitutively expressed. The constitutively expressed cancer associated antigen (e.g., neoantigen) may be present in repeat and adapted for delivery via multiple distinct Gram-negative bacterial transport systems. Examples of contemplated constitutive promoters are known to those skilled in the art and include, but are not limited to PagC, Ssph2, sicA (SEQ ID NO 69), pLac, J23105, J23119, J23109 promoters.

[0142] Embodiments consistent with the present disclosure provide a regulating expression cassette encoding a prokaryotic transcription regulator. The regulating expression cassette may be heterologous or non-native to the bacterium. The regulating expression cassette may be exogenously induced, meaning that the inducer is administered to the subject or bacteria from outside the environment of the bacteria or tumor. Outside the environment may refer to a systemically applied administration route in a subject such as by intravenous or oral route. The induction may be indirect, or direct. The regulation may be positive or negative. Typically, the inducer is administered parenterally or orally after a period of time from administration of the bacterium to allow for tumor-homing in the subject. Despite the bacteria having homed to the tumor, a safe-dose of aspirin administered to a subject is sufficient to induce the prokaryotic transcription regulator.

[0143] In a preferred embodiment, the heterologous positive regulator is SalR or NahR. As illustrated in FIG. 2, SalR is a typical LysRtype transcriptional regulator (LTTR) family protein and activates the Psal promoter when acetylsalicylic acid (ASA) or salicylate is present in the range of 0.05 to 10 microM.

[0144] In some embodiments, the exogenously inducible transcription regulator is in cis position. In some embodiments, the exogenously inducible transcription regulator is in a trans position relative to one or more inducible prokaryotic expression cassettes encoding heterologous immunomodulators.

[0145] In various embodiments, the tumor-homing bacterium is adapted to colonize a tumor for more than 18 days. In various embodiments, the tumor-homing bacteria is adapted to provide induced expression and / or secretion of a peptide after 18 days

[0146] In some embodiments of the present invention, there is provided tumor-homing bacterium, genetically modified for exogenously inducible immunomodulation. The tumorhoming bacterium may be genetically modified for exogenously inducible immunomodulation using a regulating expression cassette and an inducible DNA construct or prokaryotic expression cassette.

[0147] As described, the inducible prokaryotic expression cassette may include: a heterologous regulator-dependent prokaryotic promoter associated with a polynucleotide encoding a LIGHT peptide, said LIGHT peptide being either the whole LIGHT protein, an extracellular domain (or soluble domain) of LIGHT or a portion thereof, and being associated with a polynucleotide encoding a transport signal. In a preferred embodiment, the regulator's activity, is dependent on a salicylate or aspirin-based induction. For example, the inducible transcription regulator may be salR or nahR. In some embodiments, upon induction, the complete protein or a portion of the extracellular domain of LIGHT is present in a biologically active form. The biologically active form may be delivered specifically to the tumor. The presence of biologically active LIGHT may be measured in terms of antibody staining of the bacteria culture or of the interstitial fluid of tumors colonized by LIGHT expressing bacteria. Said another way, the peptide may undergo active or passive secretion. A biologically effective form may be measured by a LIGHT receptor binding assay or an alternate assay which assesses LIGHT activity.

[0148] In some embodiments, the heterologous immunomodulator is a fusion peptide further comprising a transport signal being a secretion signal. In some embodiments, the heterologous immunomodulator is a fusion peptide further comprising a transport signal being a wall display signal. In some embodiments, the LIGHT peptide is inserted at the adl, ttrA or Stm3120 locus or homology thereof. In some embodiments, the LIGHT peptide is inserted at the adl locus.

[0149] In some embodiments, the bacteria is genetically modified to include a regulating expression cassette encoding a heterologous, exogenously induced inducible prokaryotic transcription regulator; and an inducible prokaryotic expression cassette including: a heterologous regulator-dependent prokaryotic promoter associated with a polynucleotide encoding a GMCSF peptide, said polynucleotide being associated with a polynucleotide encoding a transport signal. In a preferred embodiment, the regulator activity, is dependent on aspirinbased induction. For example, the inducible transcription regulator may be salR or nahR. In some embodiments, upon induction such as by aspirin, the GMCSF peptide is present in a biologically active form. The biologically active form may be delivered specifically to the tumor. Thepresence of biologically active GMCSF may be measured in terms of expression, (i.e., presence of a peptide) or a presence in the or tumor environment due to secretion from the bacteria. Said another way, the peptide may undergo active or passive secretion. A biologically effective form may be measured by a GMCSF receptor binding assay or an alternate assay which assesses activity of GMCSF.

[0150] In some embodiments, the regulating expression cassette encoding a heterologous, exogenously induced inducible prokaryotic transcription regulator; and an inducible prokaryotic expression cassette comprising: a heterologous regulator-dependent prokaryotic promoter associated with a polynucleotide encoding a SIRP alpha peptide, being associated with a polynucleotide encoding a transport signal. In a preferred embodiment, the regulator activity, is dependent on aspirin-based induction. For example, the inducible transcription regulator may be salR or nahR. In some embodiments, upon induction such as by aspirin, the SIRP alpha peptide is present in a biologically active form. The biologically active form may be delivered specifically to the tumor. The presence of biologically active SIRP alpha may be measured in terms of expression or a presence in the or tumor environment due to secretion from the bacteria. Said another way, the peptide may undergo active or passive secretion. A biologically effective form may be measured by a SIRP alpha receptor binding assay or an alternate assay which assesses activity of SIRP alpha.

[0151] As described, the regulating expression cassette encoding a heterologous, exogenously induced inducible prokaryotic transcription regulator; and an inducible prokaryotic expression cassette comprising: a heterologous regulator-dependent prokaryotic promoter associated with a polynucleotide encoding a IL18 peptide, being associated with a polynucleotide encoding a transport signal. In a preferred embodiment, the regulator activity, is dependent on aspirin-based induction. For example, the inducible transcription regulator may be salR or nahR. In some embodiments, upon induction, such as by aspirin, the IL 18 peptide is present in a biologically active form. The biologically active form may be delivered specifically to the tumor. The presence of biologically active IL18 may be measured in terms of expression or a presence in the or tumor environment due to secretion from the bacteria. Said another way, the peptide may undergo active or passive secretion. A biologically effective form may be measured by a IL 18 receptor binding assay or an alternate assay which assesses activity of IL 18.

[0152] In some embodiments, the inducible prokaryotic expression cassette is inserted at the adl, ttrA, or Stm3120 locus. In some embodiments, the immunomodulator expression cassette isinserted at the adl locus or homology thereof. In some embodiments, the immunomodulator expression cassette is inserted at the Stm3120 locus or homology thereof. In some embodiments, the immunomodulator expression cassette is inserted at the ttrA locus or homology thereof.

[0153] In some embodiments, the immunomodulator is selected from a list consisting of: LIGHT, GMCSF, SIRP alpha, and IL18. In some embodiments, the immunomodulator is a LIGHT peptide. In some embodiments, upon induction, the heterologous immunomodulators are secreted and / or expressed in a biologically active form. In some embodiments, the heterologous immunomodulator is secreted and / or expressed in a biologically active form upon at least partial tumor colonization in a subject and systemic exposure to a safe dose of a salicylate, salicylic acid or derivative thereof (e.g., acetylsalicylic acid). In some embodiments, the bacterium provides a therapeutic effect on a tumor upon at least partial tumor colonization in a subject and administration of a safe dose of a salicylate, salicylic acid or derivative thereof (e.g., acetylsalicylic acid).

[0154] In some embodiments, the genetically modified bacteria comprises multiple copies of polynucleotide encoding the same immunomodulator.

[0155] In some embodiments, the heterologous immunomodulator is a LIGHT peptide. In some embodiments, the LIGHT peptide is inserted at the adl locus.

[0156] Various LIGHT peptides are known in the art. In some embodiments, the whole LIGHT peptide or a portion (i.e., peptide portion) of an extracellular or soluble domain of LIGHT is employed. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO 18 or SEQ ID NO 19. Examples of polynucleotides encoding the LIGHT peptides include but are not limited to SEQ ID NO 20 or SEQ ID NO 21.

[0157] The LIGHT peptide may be associated with a Type II or a Type V secretion signal. In a preferred embodiments, the LIGHT peptide is associated with a Type II secretion signal. In a preferred embodiments, the LIGHT peptide is fused with a Type II secretion signal such as PelB. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding polynucleotide sequence(s) having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with PelB SEQ ID NO 22.

[0158] In some embodiments, the immunomodulator is a SIRP alpha peptide. Various SIRP alpha peptides are known in the art. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SIPR alpha polypeptide SEQ ID NOs 23-26 or SIPR alpha polynucleotide SEQ ID NO 27.

[0159] The SIRP alpha peptide may be associated with a Type II or a Type V secretion signal. The SIRP alpha peptide may be associated with a Type II secretion signal such as OmpA. The SIRP alpha peptide may be fused with a Type II secretion signal such as OmpA. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding polypeptide sequence(s) having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with OmpA SEQ ID NO 28.

[0160] In some embodiments, the one or more inducible prokaryotic expression cassettes includes an immunomodulator expression cassette having an IL 18 peptide.

[0161] In some embodiments, the immunomodulator is an IL 18 peptide. Various IL 18 peptides are known in the art. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding polypeptide sequence(s) having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with IL 18 SEQ ID NO 29 , IL 18 SEQ ID NO 30, IL 18 SEQ ID NO 31 , IL 18 SEQ ID NO 32, IL18 SEQ ID NO 33, IL18 SEQ ID NO 34. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with IL18 SEQ ID NO 35, IL18 SEQ ID NO 36, IL18 SEQ ID NO 37.

[0162] The IL 18 peptide may be associated with a Type II, Type III, or a Type V secretion signal. The IL18 peptide may be associated with a Type III, or a Type V secretion signal. In preferred embodiments, the polynucleotide encoding the IL18 peptide may be associated with a polynucleotide encoding a secretion signal from a Type III secretion system. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding polypeptide(s) comprising N-terminal having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with PET protein SEQ ID NO 38. The IL18 peptide may be associated with a Type III, or a Type V secretion signal. In some embodiments, the genetically engineered bacteria comprise one or moregene sequence(s) encoding polypeptide sequence(s) comprising C-terminal having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with PET protein SEQ ID NO 39. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) encoding polypeptide(s) comprising N-terminal having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with MISSSSIS (SEQ ID NO 14) or equivalent variations disclosed in S. A. Lloyd, et.al (2002)

[0163] In some embodiments, the one or more inducible prokaryotic expression cassette includes a heterologous immunomodulator expression cassette having an GMCSF peptide. In some embodiments, the heterologous immunomodulator is a GMCSF peptide. Various GMCSF peptides are known in the art In some embodiments, the immunomodulator is a GMCSF peptide. In some embodiments, the genetically engineered bacteria comprise one or more gene sequence(s) having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with GMCSF polypeptide SEQ ID NO 40 or SEQ ID NO 41 , or GMCSF polynucleotide SEQ ID NO 42.

[0164] The GMCSF peptide may be associated with a Type II or a Type V secretion signal. The GMCSF peptide may be associated with a Type II secretion signal. The GMCSF peptide may be fused with a Type II secretion signal. Examples include but are not limited to: OmpA, pelB and preferably OmpA. In some embodiments, the genetically engineered bacteria comprise one or more polypeptide sequence(s) having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with OmpA SEQ ID NO 28.

[0165] In some embodiments, the tumor-homing bacteria is adapted to colonize a tumor for more than 18 days. In some embodiments, the tumor-homing bacteria is adapted to provide induced expression and / or secretion of a peptide after 18 days.

[0166] In some embodiments, the one or more inducible prokaryotic expression cassettes comprise a polynucleotide encoding two or more heterologous immunomodulators selected from the group consisting of: a SIRP alpha, GMCSF, LIGHT, and IL18.

[0167] In some embodiments, the immunomodulator expression cassette includes a polynucleotide encoding two or more heterologous immunomodulators selected from the groupconsisting of: a SIRP alpha, GMCSF, LIGHT, and IL18. In other embodiments, the two or more heterologous immunomodulators are on two different inducible prokaryotic expression cassettes.

[0168] In some embodiments, the one or more inducible prokaryotic expression cassettes includes a polynucleotide encoding two or more heterologous immunomodulators selected from the group consisting of: a SIRP alpha, GMCSF, LIGHT, and IL18.

[0169] In some embodiments, immunomodulator expression cassette includes a polynucleotide encoding three or more heterologous immunomodulators selected from the group consisting of: a SIRP alpha, GMCSF, LIGHT, and IL 18. In other embodiments, the three or more heterologous immunomodulators are on two or three different inducible prokaryotic expression cassettes.

[0170] In some embodiments, the following combination heterologous immunomodulators are preferred: a SIRP alpha and a GMCSF peptide; a SIRP alpha and a LIGHT peptide; a SIRP alpha, a LIGHT and a GMCSF peptide; a IL 18 and a LIGHT peptide; a SIRP alpha and a IL 18 peptide; a SIRP alpha, a IL 18 and a LIGHT peptide; a LIGHT and a GMCSF and; a SIRP alpha, a IL18, a LIGHT and a GMCSF peptide.

[0171] In some embodiments, a homologous copy of the same heterologous immunomodulator is encoded within the bacterium.

[0172] In some embodiments, the bacterium is further genetically modified to co-express two homologous cancer antigen, each respective cancer antigen peptide being fused to a transport signal from a distinct bacterial transport system. In some embodiments, the distinct bacterial transport systems are both secretion systems selected from a list consisting of: type II secretion system, type III secretion system, type V secretion signal. In some embodiments, the distinct bacterial transport systems are from type III secretion system and a type V secretion signal. In some embodiments, the two homologous cancer antigens are neoantigens.

[0173] In various embodiments described above, the recombinant bacteria is an attenuated bacteria. In some embodiments, the attenuated bacteria strain is selected from the list consisting of: Salmonella spp., Yersinia spp., Bordetella spp., Escherichia coli, Shigella spp., Burkholderia mallei, Burkholderia pseudomallei and Pseudomonas aeruginosa. In some embodiments, the bacterium is selected from a genus being a Salmonella and / or a Pseudomonas. In some embodiments, the bacterium is a Salmonella Typhimurium. In some embodiments, the expression cassettes are in an auto replicative vector (plasmid). In some embodiments, the expression cassettes are chromosomally integrated.

[0174] Embodiments consistent with the present disclosure also provide vaccines, genetically modified bacteria, and methods of treatment using bacteria genetically modified for multimodal transport of heterologous cancer-associated antigens (e.g., neoantigens) and externally controlled tumor-local delivery of immunomodulators. In this case, the genetically modified bacteria may serve any one or more of the following: 1) as an oncolytic agent leading to tumor cell death, while stimulating the immune system 2) as a targeting vehicle - homing to the tumor site to deliver a immunomodulating payloads.

[0175] In certain aspects, the recombinant bacteria are genetically modified for tumor-local delivery of one or more immunomodulators. Thus, in certain embodiments, the genetically modified tumor-homing bacteria are administered systemically and selectively home to a tumor or a tumor microenvironment. As used herein, administered systemically, refers to administration to the bloodstream where it is circulated systemically.

[0176] Interestingly, a single time exposure to aspirin was sufficient to provide a degree of tumor reduction, especially when a battery of heterologous immunomodulators were employed.

[0177] In various embodiments, the tumor-homing bacteria may encode two or more heterologous immunomodulator selected from the group consisting of: a SIRP alpha, GMCSF, LIGHT, and IL18. In various embodiments, the tumor-homing bacteria may encode three or more heterologous immunomodulator selected from the group consisting of: a SIRP alpha, GMCSF, LIGHT, and IL 18. The heterologous immunomodulator may be present in a polycistronic vector or in multiple inducible prokaryotic expression cassette. The tumor-homing bacterium may secrete and / or express any one, two, three or four of SIRP alpha, GMCSF, LIGHT, and IL 18 in a biologically active form.

[0178] In some embodiments, the tumor-homing bacterium may encode and express or secrete a set of heterologous immunomodulator selected from the group consisting of: (i) a SIRP alpha and a GMCSF, (ii) a SIRP alpha and a LIGHT, (iii) a SIRP alpha, a LIGHT and a GMCSF (iv) a IL 18 and a LIGHT, (v) a SIRP alpha and a IL 18 (v) a SIRP alpha, a IL 18 and a LIGHT, (vi) a LIGHT and a GMCSF and (vii) a SIRP alpha, a IL 18, a LIGHT and a GMCSF in a biologically active form.

[0179] In some embodiments, the tumor-homing bacterium may encode and express or secrete a SIRP alpha and a GMCSF in a biologically active form.

[0180] In some embodiments, the heterologous immunomodulator peptides are a SIRP alpha and a LIGHT. In some embodiments, the heterologous immunomodulator peptides are a SIRPalpha, a LIGHT and a GMCSF. In some embodiments, the heterologous immunomodulator peptides are a IL 18 and a LIGHT. In some embodiments, the heterologous immunomodulator peptides are a SIRP alpha and a IL 18. In some embodiments, the heterologous immunomodulator peptides are a SIRP alpha, a IL 18 and a LIGHT. In some embodiments, the heterologous immunomodulator peptides are a LIGHT and a GMCSF. In some embodiments, the heterologous immunomodulator peptides are a SIRP alpha, a IL18, a LIGHT and a GMCSF.

[0181] In some embodiments, an inducible prokaryotic expression cassette comprises a polynucleotide series encoding heterologous immunomodulator peptides being a SIRP alpha and a GMCSF. In some embodiments, a single inducible prokaryotic expression cassette comprises a polynucleotide series encoding heterologous immunomodulator peptides being a SIRP alpha and a LIGHT.

[0182] In some embodiments, an inducible prokaryotic expression cassette comprises a polynucleotide series encoding heterologous immunomodulator peptides being a SIRP alpha, a LIGHT and a GMCSF. In some embodiments, a single inducible prokaryotic expression cassette comprises a polynucleotide series encoding heterologous immunomodulator peptides being a IL 18 and a LIGHT.

[0183] In some embodiments, an inducible prokaryotic expression cassette comprises a polynucleotide series encoding heterologous immunomodulator peptides being a SIRP alpha and a IL18. In some embodiments, an inducible prokaryotic expression cassette comprises a polynucleotide series encoding heterologous immunomodulator peptides being a SIRP alpha, a IL 18 and a LIGHT.

[0184] In some embodiments, an inducible prokaryotic expression cassette comprises a polynucleotide series encoding heterologous immunomodulator peptides being a SIRP alpha and a GMCSF. In some embodiments, the prokaryotic expression cassette is inserted at the site of the deleted adl or Stm3120 locus or homology thereof.

[0185] In some embodiments, the prokaryotic expression cassette is inserted at the site of the deleted Stm3120 locus, and the series further includes a LIGHT.

[0186] In some embodiments, the prokaryotic expression cassette comprising SIRP alpha and GMCSF are inserted at the site of the deleted Stm3120 locus, and a third prokaryotic expression cassette comprises a LIGHT and is inserted at the site of the deleted adl locus. In some embodiments, the prokaryotic expression cassette comprises a series of heterologous immunomodulator peptides being SIRP alpha and LIGHT.

[0187] In some embodiments, the prokaryotic expression cassette comprises a series of heterologous immunomodulators being SIRP alpha, LIGHT and GMCSF. In some embodiments, the prokaryotic expression cassette comprises a series of heterologous immunomodulator peptides being IL 18 and LIGHT.

[0188] In some embodiments, the prokaryotic expression cassette comprises a series of heterologous immunomodulator peptides being SIRP alpha and IL 18. In some embodiments, the prokaryotic expression cassette comprises a series of heterologous immunomodulator peptides being SIRP alpha, IL 18 and LIGHT, In some embodiments, the prokaryotic expression cassette comprises a series of heterologous immunomodulator peptides being SIRP alpha, IL 18, LIGHT and GMCSF. In some embodiments, the prokaryotic expression cassette is inserted at the site of a deleted locus selected from any one of the following loci: aac6, ttrA, adl, or stm3120 loci. In some embodiments, the prokaryotic expression cassette comprising SIRP alpha, LIGHT and GMCSF is inserted at any one of the following loci: aac6, Stm3120, ttrA or adl. In some embodiments, the prokaryotic expression cassette encoding SIRP alpha, LIGHT and GMCSF is inserted at the site of the deleted ttrA locus and the third prokaryotic expression cassette encoding IL 18 is inserted at the site of the deleted adl. In some embodiments, the prokaryotic expression cassette comprising SIRP alpha, LIGHT and GMCSF is inserted at the site of the deleted STM3120 locus. In some embodiments, the prokaryotic expression cassette comprising SIRP alpha, LIGHT and GMCSF is inserted at the site of the deleted adl locus. In some embodiments, the prokaryotic expression cassette comprising SIRP alpha, LIGHT, GMCSF and IL18 is inserted at the site of the deleted adl locus. In some embodiments, the prokaryotic expression cassette comprising SIRP alpha, LIGHT, GMCSF is inserted at the site of the deleted adl locus and the prokaryotic expression cassette comprising IL 18 is inserted at the site of the deleted STM3120 locus. In some embodiments, the prokaryotic expression cassette comprising the SIRP alpha and IL 18 is inserted at the site of the deleted Stm3120 locus and the prokaryotic expression cassette comprising the LIGHT is inserted at the site of the deleted adl locus. In some embodiments, the prokaryotic expression cassette comprising the SIRP alpha, IL 18 and LIGHT is inserted at the site of the deleted adl locus.

[0189] In some embodiments, the prokaryotic expression cassette is inserted at the site of a deleted locus selected from any one of the following loci: aac6, ttrA, adl, or STM3120 loci. In some embodiments, the prokaryotic expression cassette comprising SIRP alpha, LIGHT and GMCSF is inserted at any one of the following loci: aac6, ttrA, adl or STM3120 loci. In someembodiments, the prokaryotic expression cassette encoding SIRP alpha, LIGHT and GMCSF is inserted at the site of the deleted aac6 or ttrA locus and the third prokaryotic expression cassette encoding IL18 is inserted at the site of the deleted adl. In some embodiments, the prokaryotic expression cassette comprising SIRP alpha, LIGHT and GMCSF is inserted at the site of the deleted STM3120 locus. In some embodiments, the prokaryotic expression cassette comprising SIRP alpha, LIGHT and GMCSF is inserted at the site of the deleted adl locus. In some embodiments, the prokaryotic expression cassette comprising SIRP alpha, LIGHT, GMCSF and IL18 is inserted at the site of the deleted adl locus. In some embodiments, the prokaryotic expression cassette comprising SIRP alpha, LIGHT, GMCSF is inserted at the site of the deleted adl locus and the prokaryotic expression cassette comprising IL 18 is inserted at the site of the deleted STM3120 locus. In some embodiments, the prokaryotic expression cassette comprising the SIRP alpha and IL18 is inserted at the site of the deleted STM3120 locus and the prokaryotic expression cassette comprising the LIGHT is inserted at the site of the deleted adl locus. In some embodiments, the prokaryotic expression cassette comprising the SIRP alpha, IL18 and LIGHT is inserted at the site of the deleted adl locus.

[0190] In another aspect, there is provided, a recombinant tumor-homing bacterium being an attenuated Gram-negative bacterium, wherein the recombinant bacterium is genetically modified to include a total of three or more prokaryotic expression cassette comprising: one or more constitutive prokaryotic expression cassettes encoding two homologous cancer antigens (e.g., neoantigen), each associated with a transport signal from a distinct transport system, an expression cassette encoding a regulator; and one or more inducible prokaryotic expression cassettes encoding a single or set of heterologous immunomodulators selected from a list consisting of: LIGHT, GMCSF, SIRP alpha, and IL 18.

[0191] In another aspect, there is provided, a recombinant tumor-homing bacteria, genetically modified to include three prokaryotic expression cassette including: a prokaryotic expression cassette encoding a cancer associated fusion peptide such as a neoantigen fusion peptide comprising a single or series of neoantigens and a first transport signal from a first transport system; and a prokaryotic expression cassette encoding a neoantigen fusion peptide comprising a single or series of neoantigens and a second transport signal from a second transport system; and a prokaryotic expression cassette encoding a regulator; and a prokaryotic expression cassette encoding a regulator-dependent prokaryotic promoter associated with an immunomodulator expression cassette comprising a single or series of heterologousimmunomodulators selected from the list consisting of: LIGHT, GMCSF, SIRP alpha, CD40L, and IL18. In one embodiment, the tumor-homing bacteria is modified to provide an expression or secretion of a biologically active form of the heterologous immunomodulator(s). In another embodiments, the tumor-homing bacteria is modified to provide, upon at least partial tumor colonization and administration of a subject-safe dose of a salicylic acid (e.g., acetylsalicylic acid), a measurable therapeutic effect on the tumor. A measurable therapeutic effect may include a change in immune profile of a tumor in a subject consistent with a therapeutic effect. In some embodiments, the first and second transport signal are from a Type III or Type V secretion system.

[0192] In a more particular aspect, there is provided, a tumor-homing bacterium, genetically modified for inducible tumor-local delivery of multiple heterologous immunomodulators, said tumor-homing bacterium being an attenuated Gram-negative bacterium and having a total of three or more prokaryotic expression cassette comprising: i. one or more constitutive prokaryotic expression cassettes encoding two homologous neoantigens, each associated with a transport signal from distinct transport systems, ii. a regulating expression cassette encoding a regulator; and iii. one or more aspirin-inducible prokaryotic expression cassettes encoding a set of immunomodulator fusion peptides, each of said immunomodulator fusion peptides comprising a heterologous immunomodulator associated with a secretion signal, said heterologous immunomodulator selected from a list consisting of: LIGHT, GMCSF, SIRP alpha, and IL 18.

[0193] In a particular embodiment, an illustration of the Salmonella Typhimurium genome and its modifications are presented. In FIG. 3A, STM3120 gene is deleted, aadA gene is deleted and replaced by salR regulator CDS, aac6 gene is deleted and replaced by 2 repeats of neoantigens (NeoAg) with secretion signals (T3SS - type 3, T5SS - type 5) , a neoantigen CDS is inserted into the ompA CDS in frame to obtain a fusion protein presenting the neoantigen on the bacterial cell wall, adl gene is deleted and replaced by an expression cassette with pSal promoter and 4 payloads (SIRPa, GM-CSF, IL18, and LIGHT). In FIG. 3B, aadA gene is deleted and replaced by salR regulator CDS, aac6 gene is deleted and replaced by 2 repeats of neoantigens (NeoAg) with secretion signals (T3SS - type 3, T5SS - type 5), a neoantigen CDS is inserted into the ompA CDS in frame to obtain a fusion protein presenting the neoantigen on the bacterial cell wall, adl gene is deleted and replaced by an expression cassette with pSal promoter and IL 18 CDS, STM3120 is deleted and replaced by an expression cassette with pSal promoter and 3 payloads (GM-CSF, SIRPa, and LIGHT).

[0194] In some embodiments, upon induction, the heterologous immunomodulator peptide is secreted and / or expressed in a biologically active form.

[0195] In some embodiments, the bacteria has no deactivating mutation in an outer membrane protein; and wherein each of the respective immunomodulator fusion peptides are associated with a secretion signal from a Type V secretion system or an inner membrane secretion system (e.g., sec or tat secretion system).

[0196] In some embodiments, the regulating expression cassette encoding a regulator is either i. inserted at the aadA locus (i.e., rendering a deactivating mutation in the aadA gene); or ii. is in a trans position relative to the one or more inducible prokaryotic expression cassettes and wherein the inducible promoter is induced by a salicylic acid or derivative thereof. In some embodiments, the regulating expression cassette encoding a regulator is inserted at the aadA locus is a cis position.

[0197] In some embodiments, the inducible prokaryotic expression cassette is a polycistronic vector comprising two or more polynucleotide sequences selected from the list of: LIGHT, SIRP alpha, GMCSF and IL 18. In some embodiments, a ratio of the number of inducible prokaryotic expression cassettes to the regulating expression cassette is 2: 1 or more.

[0198] In some embodiments, the regulating expression cassette encoding a regulator is in trans position relative to the one or more inducible prokaryotic expression cassettes.

[0199] In some embodiments, the inducible promoter is induced by a small molecule selected from the list consisting of L-arabinose, IPTG, or a salicylic acid, acetylsalicylic acid or derivative thereof. In some embodiments, the inducible prokaryotic expression cassette is inserted at the adl, ttrA, or Stm3120 locus.

[0200] In some embodiments, the LIGHT peptide is inserted at the adl locus. In some embodiments, the one or more inducible prokaryotic expression cassettes comprises an immunomodulator expression cassette having an IL 18 peptide. In some embodiments, the one or more inducible prokaryotic expression cassettes comprises an immunomodulator expression cassette having an GMCSF peptide. In some embodiments, the one or more inducible prokaryotic expression cassettes comprise a polynucleotide encoding two or more heterologous immunomodulators selected from the group consisting of: a SIRP alpha, GMCSF, LIGHT, and IL 18. In some embodiments, one or more inducible prokaryotic expression cassettes comprises a polynucleotide encoding three or more heterologous immunomodulator selected from the group consisting of: a SIRP alpha, GMCSF, LIGHT, and IL 18.

[0201] In some embodiments, the tumor-homing bacterium is an attenuated bacteria is selected from the list consisting of: Salmonella spp., Yersinia spp., Bordetella spp., Escherichia coli, Shigella spp., Burkholderia mallei, Burkholderia pseudomallei and Pseudomonas aeruginosa. In some embodiments, the bacteria is selected from a genus being a Salmonella and / or a Pseudomonas. In some embodiments, the bacterium is a Salmonella Typhimurium.

[0202] In some embodiments, the expression cassettes are in an auto replicative vector (plasmid). In an alternative embodiment, the expression cassettes are chromosomally integrated.

[0203] In another aspect, there is provided, a vaccine comprising a bacterium according to any of the above and one or more pharmaceutically acceptable carriers or excipients.

[0204] In another aspect, there is provided, a method of treating a cancer of a subject in need thereof comprising administering to the subject an effective amount of the vaccine, thereby treating the cancer. In some embodiments, subsequent to administering a dose of bacteria, upon tumor-colonization of the bacteria in a cancer tumor and / or a reduction in a systemic bacterial load in a subject; and administering a salicylate, salicylic acid or derivative thereof. In some embodiments, the administering of an inducer is by a single or preferably periodic administration. In some embodiments, administering the bacteria is systemically exposing the subject to the bacterium. In some embodiments, the administering to the subject is by parenteral administration. In some embodiments, the administering of the bacteria is by intratumoral route. In some embodiments, the administering to the subject is by oral administration.

[0205] In some embodiments, the cancer is selected from the group consisting of breast, melanoma, colorectal cancer, lung cancer, gastric cancer, pancreatic cancer, ovarian cancer, bone cancer and brain cancer.

[0206] In some embodiments, the upon induction, the heterologous immunomodulator is secreted and / or expressed in a biologically active form.

[0207] In another aspect, there is provided, a vaccine comprising a bacterium of the present disclosure and an excipient or carrier. In another aspect, there is provided, a method of treating a cancer of a subject in need thereof comprising administering to the subject an effective amount of the vaccine, thereby treating the cancer or providing adjuvant or neoadjuvant therapy.

[0208] Treating a cancer may involve inducing an antigen specific immune response against the cancer associated antigen or neoantigen when administered to a subject in need thereof, (e.g., a CD8+ cellular immune response). Alternatively, treating a cancer may involve recruitment of anti-tumor cytotoxic T cells, neutralizing immune suppressive cells, or activation of exhaustedtumor microenvironment immune cells. Treating a cancer may involve reducing a tumor volume. Treating a cancer may involve reducing a solid tumor volume.

[0209] In some embodiments, the cancer is breast, melanoma, colorectal cancer, lung cancer, gastric cancer, pancreatic cancer, ovarian cancer, bone cancer and brain cancer.

[0210] In some embodiments, subsequent to administering a dose of bacteria, upon tumorcolonization of the bacteria in a cancer tumor and / or a reduction in a systemic bacterial load, administering an inducer. In some embodiments, the inducer is a salicylate or salicylic acid.

[0211] In some embodiments, administering an inducer is a by periodic administration. In some embodiments, administering an inducer is readministering a salicylate or salicylic acid after a period of time without immunomodulator expression or secretion. In some embodiments, wherein administering an inducer at least 18 days after administering a dose of bacteria provides induced expression and / or secretion of a peptide. In some embodiments, induction is provided after day 18, post administration. In some embodiments, administering to the subject provides systemic exposure of the vaccine.

[0212] In some embodiments, administering to the subject is by parenteral administration (e.g., injection or intravenous infusion). In some embodiments, administering to the subject is by oral administration.

[0213] In some embodiments, the effective amount of bacteria is administered as adjuvant therapy post tumor resection.

[0214] In various embodiments, the tumor-homing bacterium is adapted to provide induced expression and / or secretion of a peptide after 18 days. In various embodiments, the heterologous immunomodulator is exogenously induced, for example by administering a salicylic acid, acetylsalicylic acid or derivatives thereof.

[0215] In various embodiments, the tumor-homing bacterium is adapted for a single efficacious induction. Single efficacious induction refers to a single time exposure of the tumor-homing bacterium that encodes an immunomodulator fusion peptide to an inducer which results in an extent of tumor reduction.

[0216] Thus, in another aspect of the present invention, there is provided a method of treating a cancer in a subject in need thereof comprising: i. administering a Gram-negative tumorhoming bacterium to a subject in need thereof, said Gram negative bacteria genetically modified for aspirin inducible expression of immunomodulator fusion peptide, said immunomodulatorfusion peptide comprising a heterologous immunomodulator characterized by a 1 to 4% cysteine content and having a length of between 30 to 400 amino acids and being linked to an inner membrane secretion signal, such as the Sec or tat secretion system; upon tumor-colonization of the bacteria in a cancer tumor and / or reduction of a systemic bacterial load in a subject, administering to the subject a safe dose of aspirin. Consistent with some embodiments of the present disclosure, administering is systemically administering, such as by intravenous route. Treating a cancer may refer to providing a measurable reduction in tumor volume. Consistent with some embodiments of the present disclosure, the Gram-negative tumor-homing bacterium is selected from a list consisting of: Salmonella spp., Yersinia spp., Bordetella spp., Escherichia coli, Shigella spp., Burkholderia mallei, Burkholderia pseudomallei and Pseudomonas aeruginosa. In preferred embodiments, the bacteria is Salmonella or specifically Salmonella Typhimurium. In some embodiments, a heterologous immunomodulator characterized by a 1 to 4% cysteine content and having a length of between 30 to 400 amino acids may be LIGHT, GMCSF, or SIRP alpha. In some embodiments, the heterologous immunomodulator may have a hydrophobicity range of between 0.5 to 0.01 on the Kyte-Doolittle hydrophobicity index. Consistent with some embodiments, administering to the subject a safe dose of aspirin results in presence (e.g., via secretion) of an anti-tumor effective dose of biologically active heterologous immunomodulator in the tumor environment.

[0217] In another aspect of the present invention, there is provided a method of treating a cancer in a subject in need thereof comprising: i. administering a Gram-negative tumor-homing bacterium to a subject in need thereof, said Gram negative bacteria genetically modified for aspirin inducible expression of an IL 18 fusion peptide comprising an IL 18 linked to a Type III signal sequence; upon tumor-colonization of the bacteria in a cancer tumor and / or reduction of a systemic bacterial load in a subject, and administering to the subject a safe dose of aspirin. Consistent with some embodiments of the present disclosure, administering the Gram-negative tumor-homing bacterium is systemically administering, such as by intravenous route, while administering the aspirin is by oral route. Type III signal sequences are known in the art and include, for example, MISSSSIS (SEQ ID NO 43). Treating a cancer may refer to providing a measurable reduction in tumor volume. Consistent with some embodiments of the present disclosure, the Gram-negative tumor-homing bacterium is selected from a list consisting of: Salmonella spp., Yersinia spp., Bordetella spp., Escherichia coli, Shigella spp., Burkholderia mallei, Burkholderia pseudomallei and Pseudomonas aeruginosa. In preferred embodiments, thebacteria is Salmonella or specifically Salmonella Typhimurium. Consistent with some embodiments, administering to the subject a safe dose of aspirin results in presence (e.g., via secretion) of an anti-tumor effective dose of biologically active IL188 in the tumor environment

[0218] Consistent with some embodiments of the present disclosure, there is provided a method of treating cancer in a subject in need thereof comprising administering to the subject an effective amount of the bacterial strain or vaccine, thereby treating the cancer. The vaccine in this context relates to any formulation of recombinant tumor-homing bacterium described herein. As used herein, treating cancer in a subject involves reducing a tumor volume and especially reducing a solid tumor volume or extending disease free survival or overall survival.

[0219] In various embodiments, the tumor-homing bacterium is adapted for a single efficacious induction. Single efficacious induction refers to a single time exposure of the tumor-homing bacterium that encodes an immunomodulator fusion peptide to an inducer which results in an extent of tumor reduction.

[0220] In some embodiments, subsequent to administering a dose of bacteria, upon tumorcolonization of the bacteria in a cancer tumor and / or a reduction in a systemic bacterial load, administering an inducer. In some embodiments, the inducer is a salicylate or salicylic acid. In some embodiments, administering an inducer is by periodic administration. In some embodiments, administering an inducer is readministering a salicylate or salicylic acid after a pause in expression or secretion of an immunomodulator. In some embodiments, administering an inducer is after day 18, post bacterial administration. In some embodiments, administering to the subject provides a systemic exposure to the vaccine. In some embodiments, administering to the subject is by parenteral administration. In some embodiments, administering to the subject is by oral administration.

[0221] In some embodiments, the cancer is selected from the group consisting of breast, melanoma, colorectal cancer, lung cancer, gastric cancer, pancreatic cancer, ovarian cancer, bone cancer and brain cancer.

[0222] In some embodiments, administering to the subject further includes administering to the subject a second vaccine comprising a ghost bacterium, said bacteria being genetically modified to express at least one cancer associated antigen or neoantigen on a cell wall of said bacteria.

[0223] In another aspect, there is provided, a method of treating a cancer in a subject comprising: administering to a blood stream of a subject in need thereof, a tumor-homing bacterium genetically modified for constitutive multi-model secretion of two homologous cancerassociated antigen (e.g., homologous neoantigens) and inducible expression of a set of heterologous immunomodulators comprising three or more peptides from the following: SIRP alpha, GMCSF, IL-18 and LIGHT; and upon tumor-colonization of the bacteria in a cancer tumor and / or a reduction in a systemic bacterial load, administering an inducer. In some embodiments, administering to a blood stream of a subject comprises intravenous administration. In some embodiments, multi-model secretion of homologous cancer associated antigens, e.g., homologous neoantigens comprises secretion by an inner membrane secretion system or a Type V secretion system. In some embodiments, further to the multi-model secretion of homologous cancer associated antigen (e.g., homologous neoantigens), a homologous neoantigen or cancer associated antigen is displayed on the cell wall surface. In some embodiments, the tumor-homing bacterium is a Gram-negative bacterium or Gram-negative bacterial strain.

[0224] Consistent with embodiments, in another aspect, there is provided, a tumor-homing bacterium genetically modified to include three or more prokaryotic expression cassette: i. a constitutive prokaryotic expression cassette encoding two homologous cancer associated antigen (e.g., homologous neoantigens), each associated with a transport signal from a distinct transport system, ii. a regulating expression cassette encoding a regulator; and iii. an inducible prokaryotic expression cassette comprising an inducible transcription promoter and a polynucleotide encoding a single or series of heterologous immunomodulators selected from a list consisting of: LIGHT, GMCSF, SIRP alpha, and IL18, and wherein each polynucleotide encoding a heterologous immunomodulator is associated with transport signal.

[0225] Consistent with embodiments, the inducible transcription promoter and regulator are induced by a small molecule that is safe for humans, including but not limited to, L- arabinose, IPTG, or salicylic acid or derivative thereof. In preferred embodiments, the small molecule is regulated and induced by a salicylic acid, acetylsalicylic acid or derivative thereof.

[0226] Consistent with embodiments, the regulating expression cassette encoding a regulator is in trans position relative to the inducible prokaryotic expression cassette.

[0227] Consistent with embodiments, a ratio of the number of inducible prokaryotic expression cassettes to the number of regulating expression cassettes may be a 2: 1 ratio or more.

[0228] Consistent with embodiments, in a another aspect, there is provided, a tumor-homing bacterium genetically modified to include three or more prokaryotic expression cassette: i. a constitutive prokaryotic expression cassette encoding two homologous cancer associated antigen (e.g., homologous neoantigens), each associated with a transport signal from a distinct transportsystem, ii. a regulating expression cassette encoding a regulator in trans position relative to the inducible prokaryotic expression cassette or inserted within the Aminoglycoside (3") (9) adenylyl transferase (aadA) gene; and iii. an inducible prokaryotic expression cassette comprising an inducible transcription promoter and a polynucleotide encoding a heterologous immunomodulator linked to a secretion signal.

[0229] Consistent with some embodiments of this aspect, the distinct transport systems are a Type III and Type V transport system. Consistent with some embodiments of this aspect, the distinct transport systems are a Type III transport system, a Type V transport system and a surface display signal. Consistent with some embodiments of this aspect, the distinct transport systems are a Type II, Type III transport system, a Type V transport system and a surface display signal.

[0230] Consistent with some embodiments of the present disclosure, the heterologous immunomodulator is linked to a secretion signal from an inner membrane secretion signal (e.g., a sec or tat secretion system) and wherein the outer membrane of the bacterium lacks any deactivating mutations. The bacterium includes effector proteins from secretion system. For example, the bacterium may be a Gram-negative bacterium.

[0231] Consistent with some embodiments of this aspect, a single or series of heterologous immunomodulators may be selected from a list consisting of: LIGHT, GMCSF, SIRP alpha, and IL 18, and wherein each polynucleotide encoding a heterologous immunomodulator.

[0232] Consistent with some embodiments of this aspect, a ratio of the number of inducible prokaryotic expression cassettes to the number of regulating expression cassettes may be a 2: 1 ratio or more.

[0233] Consistent with some embodiments of this aspect, each heterologous immunomodulator may be between 30 and 400 amino acids long. Consistent with some embodiments of this aspect, each heterologous immunomodulator may include a % cysteine content of at least 1% or between 1 and 4%. In some embodiments, the heterologous immunomodulator may have a hydrophobicity range of between 0.5 to 0.01 on the Kyte-Doolittle hydrophobicity index.

[0234] Consistent with some embodiments of this aspect, one or a set of inducible prokaryotic expression cassette comprise multiple heterologous immunomodulators, separated by cleavage site polypeptides or wherein each respective polynucleotide encoding a heterologous immunomodulator is associated with a ribosomal binding site.

[0235] Consistent with some embodiments of this aspect, the inducible transcription promoter is induced by a salicylic acid, acetylsalicylic acid or derivative thereof.

[0236] In various embodiments, the bacterium is adapted to, upon induction, secrete and / or express a light peptide in a biologically active form. In some embodiments, the bacterium is adapted to, upon tumor colonization and induction, secrete and / or express a LIGHT peptide in a biologically active form within the tumor environment. In some embodiments, the inducible prokaryotic expression cassette comprises a polynucleotide encoding a full LIGHT peptide, a portion of a LIGHT peptide such as the extracellular domain of LIGHT. The polynucleotide encoding a LIGHT peptide may be associated with a transport signal from an inner membrane secretion system (e.g., sec or tat secretion system) wherein the outer membrane of the bacterium lacks any deactivating mutations. Additionally or alternatively, the polynucleotide encoding a LIGHT peptide may be associated with a transport signal from an inner membrane secretion system (e.g., sec or tat secretion system) while the bacterium includes effector proteins from the sec / tat system, such as a Gram-negative bacterium.

[0237] In various embodiments, an inducible prokaryotic expression cassette(s) further comprises a polynucleotide encoding an IL 18 peptide. In some embodiments, two different inducible prokaryotic expression cassettes comprise the polynucleotide encoding an IL 18 peptide and the polynucleotide encoding a LIGHT peptide. In some embodiments, a single inducible prokaryotic expression cassette includes a polynucleotide encoding an IL 18 peptide and a LIGHT peptide. In some embodiments, a polynucleotide encoding a IL18 peptide is associated with a polynucleotide encoding a secretion signal from a Type V secretion system. In some embodiments, an inducible prokaryotic expression cassette(s) further comprises a polynucleotide encoding a GMCSF peptide. In some embodiments, two different inducible prokaryotic expression cassettes comprise a polynucleotide encoding a IL 18 peptide, a polynucleotide encoding a light peptide and a polynucleotide encoding the GMCSF peptide. In some embodiments, a single inducible prokaryotic expression cassette includes a polynucleotide encoding an IL 18 peptide, a LIGHT peptide and a GMCSF peptide. In some embodiments, the GMCSF peptide is associated with secretion signal from a Type II secretion system. In some embodiments, an inducible prokaryotic expression cassette(s) further comprises a polynucleotide encoding a SIRP alpha peptide. In some embodiments, two or more inducible prokaryotic expression cassettes comprise the polynucleotide encoding a IL 18 peptide, the polynucleotide encoding a light peptide, the polynucleotide encoding a GMCSF peptide and the polynucleotide encoding the SIRP alpha peptide. In some embodiments, a single inducible prokaryotic expression cassette includes a polynucleotide encoding an IL 18 peptide, a LIGHTpeptide and a SIRP alpha peptide. In some embodiments, the SIRP alpha peptide is associated with secretion signal from a Type II secretion system.

[0238] In various embodiments, an inducible prokaryotic expression cassette is inserted at an adl, ttrA, Stm3120 locus or a homology thereof. In various embodiments, multiple inducible prokaryotic expression cassettes are inserted multiple loci selected from the adl, ttrA, Stm3120 or a homology thereof. In some embodiments, at least one immunomodulator expression cassette is inserted at the adl locus or homology thereof.

[0239] In various embodiments, there is provided, a recombinant bacterium being a tumorhoming, gram-negative bacterium, genetically modified to include one or more prokaryotic expression cassette comprising: a polynucleotide encoding a first and second homologous cancer associated antigen or neoantigen polynucleotide, each encoding a single or series of cancer associated antigens or neoantigens, each of the first and second homologous cancer associated antigens or neoantigen polynucleotides being associated with a polynucleotide encoding a transport signal from a distinct transport system, said transport system being a bacterial secretion system selected from a Type III secretion system and a Type V secretion system. In some embodiments, the recombinant bacterium is genetically modified to include a third homologous cancer associated antigen or neoantigen polynucleotide associated with a polynucleotide encoding a transport signal from a distinct transport system. In some embodiments, the distinct transport system is an outer cell wall display system or a Type II secretion system. In some embodiments, each neoantigen polynucleotide is operably linked to a constitutive prokaryotic promoter. In some embodiments, each neoantigen polynucleotides encode a series of neoantigens. In some embodiments, the series of neoantigens does not include a cleavage site nor linker between each respective neoantigen. In some embodiments, the series of neoantigens includes a cleavage site or a linker between each respective neoantigen. In some embodiments, each neoantigen polynucleotide includes a different sequence of nucleotides while the encoded amino acid sequence remains the same. In some embodiments, each of the neoantigen polynucleotides encode a series of reordered neoantigens relative to the other neoantigen polynucleotide.

[0240] In various embodiments, the prokaryotic expression cassette are chromosomally integrated without negatively impacting the viability of the bacteria before administration to a subject.

[0241] In various embodiments, the recombinant bacterium is further modified to reduce virulence, reduce toxicity, reduce pathogenicity, increase tumor-homing, and / or decrease antibiotic resistance. For example, the recombinant bacterium may be further modified to include a mutation, null mutation or expression reduction of at least one gene selected from the group consisting of arginine deiminase (adl), Aminoglycoside (3") (9) adenylyl transferase (aadA), L-asparaaginase II (ansB), AAC(6’)-Iaa (aac6) and Tetrathionate reductase A (ttrA) as compared to non- attenuated bacteria of the species Salmonella enterica. In some embodiments, the recombinant bacterium is modified to include a null mutation of three of the following: stm3120, aadA, adl and aac6. In some embodiments, the recombinant bacterium is genetically modified to express a heterologous immunomodulator having direct or indirect inducible expression.

[0242] In another aspect, there is provided, a tumor-homing bacteria comprising a polynucleotide sequence encoding an IL 18 polypeptide fused to a secretion tag, wherein the polynucleotide sequence is operably linked to an aspirin-inducible promoter. In some embodiments, the secretion tag is any Type V secretion signal including but not limited to MISSSSISIS, MISSSSSSIS, or MISSSSSI. In some embodiments, the IL18 polypeptide comprises at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to any one of SEQ ID NOs 29-34 or a functional fragment thereof. In some embodiments, the secretion tag polypeptide comprises at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to any one of SEQ ID NO 43 - 45. Other relevant sequences are provided by Lloyd, S.A. et al, (2022).

[0243] In another aspect, there is provided, a tumor-homing bacteria comprising a polynucleotide sequence encoding a LIGHT polypeptide fused to a secretion tag, wherein the polynucleotide sequence is operably linked to an aspirin-inducible promoter. In some embodiments, the secretion tag is any of the Sec or Tat pathway mediated Type II secretion signals or MISSSSIS. In some embodiments, the LIGHT polypeptide comprises at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to any one of SEQ ID NOs 18-19 or a functional fragment thereof.

[0244] In another aspect, there is provided, a tumor-homing bacteria comprising a polynucleotide sequence encoding a GMCSF polypeptide fused to a secretion tag, wherein the polynucleotide sequence is operably linked to an aspirin-inducible promoter. In some embodiments, the secretion tag is Sec or Tat pathway mediated Type II secretion signal. In some embodiments, the GMCSF polypeptide comprises at least 80%, 85%, 90%, 95%, 96%, 97%,98%, or 99% identity to SEQ ID NO 40, or SEQ ID 41 or a functional fragment thereof. In some embodiments, the PelB secretion tag polypeptide comprises at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to Sequence D.

[0245] In another aspect, there is provided, a tumor-homing bacteria comprising a polynucleotide sequence encoding an SIRP alpha polypeptide fused to a secretion tag, wherein the polynucleotide sequence is operably linked to an aspirin-inducible promoter. In some embodiments, the secretion tag is any of a Sec or Tat pathway mediated Type II secretion signal. In some embodiments, the GMCSF polypeptide comprises at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO 40, or SEQ ID NO 4 lor a functional fragment thereof.

[0246] As will be readily appreciated by a skilled artisan, methods for producing the bacteria may include three main processing steps: organism banking, organism production, and preservation.

[0247] In various embodiments, there is provided, a vaccine composition comprising an excipient and any one of the bacteria described herein. Embodiments consistent with the present disclosure provide a single bacterium or population having multiple genetic modifications. In alternative embodiments, a bacterial strain may be present in a vaccine in multiple bacterial populations, each population having only a portion of the number of genetic modifications described herein. For example, a first population may be genetically modified to include the first prokaryotic expression cassette and a second population may include the second prokaryotic expression cassette, etc.

[0248] Disclosed embodiments may include any one of the following bullet-pointed features alone or in combination with one or more other bullet-pointed features, whether implemented as a system, device, and / or method.

[0249] Also disclosed herein are following clauses:

[0250] Clause 1. A tumor homing bacterium, genetically modified for inducible tumor-local delivery of multiple heterologous immunomodulators, said tumor homing bacterium being an attenuated Gram-negative bacterium and having a total of three or more prokaryotic expression cassettes comprising: i. one or more constitutive prokaryotic expression cassettes encoding two homologous cancer associated antigens (e.g., neoantigens), each associated with a transport signal from a distinct transport system, ii. a regulating expression cassette encoding a regulator; and iii. one or more inducible prokaryotic expression cassettes encoding a set of immunomodulator fusion peptides operably linked to an inducible promoter, each of saidimmunomodulator fusion peptides comprising a heterologous immunomodulator associated with a secretion signal.

[0251] Clause 2. A tumor homing bacterium, genetically modified for inducible expression of an immunomodulator including a regulating expression cassette encoding a regulator; and one or more inducible prokaryotic expression cassettes comprising: a directly or indirectly inducible promoter associated with an immunomodulator expression cassette having a polynucleotide encoding a single or series of heterologous immunomodulators peptides, wherein the inducible promoter is induced by a small molecule that is safe to administer to a subject.

[0252] Clause 3. The tumor homing bacterium of any one of clauses 1 to 2, wherein the inducible promoter and regulator are induced by a small molecule selected from the list consisting of L-arabinose, IPTG, or a salicylic acid, acetylsalicylic acid or derivative thereof.

[0253] Clause 4. The tumor homing bacterium of clause 2, wherein the inducible promoter is an aspirin-inducible promoter.

[0254] Clause 5. The tumor homing bacterium of clause 1, wherein the inducible promoter is induced by a salicylic acid or derivative thereof, and wherein the regulating expression cassette encoding a regulator is either i. inserted at the aadA locus rendering a deactivating mutation in the aadA gene; or ii. is in a trans position relative to the one or more inducible prokaryotic expression cassettes.

[0255] Clause 6. The tumor homing bacterium of clause 1, wherein the bacterium has no deactivating mutation in an outer membrane protein; wherein the inducible promoter is induced by a salicylic acid or derivative thereof, wherein the heterologous immunomodulators associated with a secretion signal which is from an inner membrane secretion system (e.g., a sec or tat secretion system).

[0256] Clause 7. The tumor homing bacterium of clause 2, wherein the bacterium has no deactivating mutation in an outer membrane protein; wherein the inducible promoter is induced by a salicylic acid or derivative thereof, and wherein the secretion signal is from an inner membrane secretion system (e.g., a sec or tat secretion system).

[0257] Clause 8. The tumor homing bacterium of clause 6 to 7, wherein the secretion signal is from a Type III secretion system.

[0258] Clause 9. The tumor homing bacterium of clause 6 to 7, wherein the secretion signal is from a sec or tat secretion system.

[0259] Clause 10. The tumor homing bacterium of any one of clauses 1 to 9, wherein the heterologous immunomodulator is selected from a list consisting of: LIGHT, GMCSF, SIRP alpha, and IL 18.

[0260] Clause 11. The tumor homing bacterium of clause 10, wherein the heterologous immunomodulator is associated with a transport signal from a distinct transport system.

[0261] Clause 12. The tumor homing bacterium of any one of clauses 1 to 11 , wherein the transport signals from distinct transport systems are transport signals from a Type III and a Type V secretion system.

[0262] Clause 13. The tumor homing bacterium of any one of clauses 1 to 12, wherein upon induction, the immunomodulator peptides are secreted and / or expressed in a biologically active form.

[0263] Clause 14. The tumor homing bacterium of any one of clauses 1 to 13, wherein induction of the tumor homing bacterium by a single and safe dose of aspirin administered to a subject, provides a tumor reducing dose of heterologous immunomodulators.

[0264] Clause 15. The tumor homing bacterium of any one of clauses 1 to 13, wherein the tumor homing bacterium is adapted to provide a therapeutic effect on a tumor upon at least partial tumor colonization in a subject and administration of a safe dose of a salicylic acid or derivative thereof.

[0265] Clause 16. The tumor homing bacterium of any one of clauses 1 to 15, wherein a ratio of the number of inducible prokaryotic expression cassettes to the regulating expression cassette is 2: 1 or more.

[0266] Clause 17. The tumor homing bacterium of any one of clauses 1 to 16, wherein the regulating expression cassette encoding a regulator is in trans position relative to the one or more inducible prokaryotic expression cassettes.

[0267] Clause 18. The tumor homing bacterium of any one of clauses 1 to 17, wherein the inducible prokaryotic expression cassette is a polycistronic vector comprising two or more, three or more or four or more polynucleotide sequences selected from the list of: LIGHT, SIRP alpha, GMCSF and IL 18.

[0268] Clause 19. The tumor homing bacterium of clause 10, wherein the immunomodulator peptide is a LIGHT peptide.

[0269] Clause 20. The tumor homing bacterium of clause 10, wherein the heterologous immunomodulator is a GMCSF peptide.

[0270] Clause 21. The tumor homing bacterium of clause 10, wherein the heterologous immunomodulator is an IL 18 peptide.

[0271] Clause 22. The tumor homing bacterium of clause 10, wherein the heterologous immunomodulator is a SIRP alpha.

[0272] Clause 23: The tumor homing bacterium bacteria of any one of clauses 1-22, wherein the strain is modified to include a deletion of any one or all of the genes: stm3120, arginine deiminase (adl), Aminoglycoside (3") (9) adenylyl transferase (aadA), and AAC(6’)-Iaa (aac6) as compared to non-attenuated bacteria of the species Salmonella enterica.

[0273] Clause 24 The tumor homing bacterium of any one of clauses 1 to 22, wherein the inducible prokaryotic expression cassette is inserted within the adl, ttrA, or Stm3120 gene.

[0274] Clause 25. The tumor homing bacterium of any one of clauses 1 to 19, wherein the LIGHT peptide is inserted at the adl locus.

[0275] Clause 26. The tumor homing bacterium of any one of clauses 1 to 18, wherein one or more inducible prokaryotic expression cassettes comprises a polynucleotide encoding two or three or more heterologous immunomodulator selected from the group consisting of: a SIRP alpha, GMCSF, LIGHT, and IL 18.

[0276] Clause 27. The tumor homing bacterium of any one of clauses 1 to 26, wherein the genetically modified tumor homing bacterium is an attenuated bacterium.

[0277] Clause 28. The tumor homing bacterium of any one of clauses 1 to 27, wherein the genetically modified tumor homing bacterium is an attenuated pathogenic bacterium.

[0278] Clause 29. The tumor homing bacterium of clause 28, wherein the attenuated pathogenic bacterium is selected from the list consisting of: Salmonella spp., Yersinia spp., Bordetella spp., Escherichia coli, Shigella spp., Burkholderia mallei, Burkholderia pseudomallei and Pseudomonas aeruginosa.

[0279] Clause 30. The tumor homing bacterium of clause 28, wherein the attenuated pathogenic bacterium is selected from a genus being a Salmonella and / or a Pseudomonas.

[0280] Clause 31. The tumor homing bacterium of clause 23, wherein the bacterium is a Salmonella Typhimurium.

[0281] Clause 32. The tumor homing bacterium of any one of clauses 1 to 31, wherein the expression cassettes are in an auto replicative vector or plasmid.

[0282] Clause 33. The tumor homing bacterium of any one of clauses 1 to 31, wherein the expression cassettes are chromosomally integrated.

[0283] Clause 34. Tumor homing bacterium, genetically modified for inducible tumor-local delivery of multiple heterologous immunomodulators, said tumor homing bacterium being attenuated bacteria having no deactivating mutations in an outer membrane and having a total of two or more prokaryotic expression cassettes comprising: i. one or more constitutive prokaryotic expression cassettes comprising two or more homologous neoantigen polynucleotides, each associated with a transport signal from a distinct transport system, and ii. an inducible prokaryotic expression cassette encoding a set of immunomodulator fusion peptides operably linked to an inducible promoter, wherein each immunomodulator fusion peptide comprises a heterologous immunomodulator associated with a secretion signal from either an inner membrane secretion system (e.g., sec or tat secretion system) or a Type III secretion system. The tumor homing bacterium includes a set of effectors proteins for functional sec or tat secretion system or a Type III secretion system.

[0284] Clause 35. The tumor homing bacterium of clause 34, wherein the inducible prokaryotic expression cassette refers to a directly or indirectly inducible promoter.

[0285] Clause 36. The tumor homing bacterium of any one of clauses 34 to 35, wherein upon induction, the heterologous immunomodulator fusion peptide is secreted and / or expressed in a biologically active form.

[0286] Clause 37. The tumor homing bacterium of any one of clauses 34 to 36, wherein the set of immunomodulator fusion peptides comprises at least cysteine-rich heterologous immunomodulator linked to a secretion signal from an inner membrane secretion system (e.g., sec or tat secretion system), said cysteine-rich heterologous immunomodulator encoding a peptide having a percent cysteine content of more than 1% or between 1 and 4% and / or hydrophobicity range of between 0.5 to 0.01 on the Kyte-Doolittle hydrophobicity index.

[0287] Clause 38. The tumor homing bacterium of any one of clause 34 to 37, wherein the set of immunomodulator fusion peptides comprises heterologous immunomodulator peptide having an amino acid length of between 30 and 400 amino acids.

[0288] Clause 39. The tumor homing bacterium of any one of clause 34 to 38, wherein the heterologous immunomodulators are selected from a list consisting of: LIGHT, GMCSF, SIRP alpha, and IL 18, and wherein the inducible promoter is induced by a small molecule selectedfrom the list consisting of L-arabinose, IPTG, or a salicylic acid, acetylsalicylic acid or derivative thereof.

[0289] Clause 40. The tumor homing bacterium of any one of clause 34 to 38, wherein the inducible promoter is an aspirin-inducible promoter.

[0290] Clause 41. The tumor homing bacterium of clause 34 to 38, wherein the inducible promoter is induced by a salicylic acid or derivative thereof, and wherein the regulating expression cassette encoding a regulator is either i. inserted at the aadA locus (i.e., rendering a deactivating mutation in the aadA gene); or ii. is in a trans position relative to the one or more inducible prokaryotic expression cassettes.

[0291] Clause 42. The tumor homing bacterium of any one of clauses 34 to 38, and 40 to 41, wherein the heterologous immunomodulator is selected from a list consisting of: LIGHT, GMCSF, SIRP alpha, and IL18.

[0292] Clause 43. The tumor homing bacterium of any one of clauses 34 to 42, wherein the transport signals from distinct transport systems are transport signals from a Type III and a Type V secretion system.

[0293] Clause 44. The tumor homing bacterium of any one of clauses 34 to 43, wherein upon induction, the heterologous immunomodulator is secreted in a biologically active form.

[0294] Clause 45. The tumor homing bacterium of any one of clauses 34 to 44, wherein induction of the tumor homing bacterium by a single and safe dose of aspirin administered to a subject, provides a tumor reducing dose of heterologous immunomodulators.

[0295] Clause 46. The tumor homing bacterium of any one of clauses 34 to 44, wherein the tumor homing bacterium is adapted to provide a therapeutic effect on a tumor upon at least partial tumor colonization in a subject and administration of a safe dose of a salicylic acid or derivative thereof.

[0296] Clause 47. The tumor homing bacterium of any one of clauses 34 to 46, wherein a ratio of the number of inducible prokaryotic expression cassettes to the regulating expression cassette is 2: 1 or more.

[0297] Clause 48. The tumor homing bacterium of any one of clauses 34 to 47, wherein the inducible prokaryotic expression cassette is a polycistronic vector comprising two or more, three or more or four or more polynucleotide sequences encoding multiple heterologous immunomodulators .

[0298] Clause 49. The tumor homing bacterium of any one of clauses 42, wherein the heterologous immunomodulator is a LIGHT peptide having more than 80% or 90% or 95% similarity to SEQ ID NOS 23-36.

[0299] Clause 50. The tumor homing bacterium of clause 42, wherein the heterologous immunomodulator is a GMCSF peptide having more than 80% or 90% or 95% similarity to SEQ ID NOS 49-51.

[0300] Clause 51. The tumor homing bacterium of clause 42, wherein the heterologous immunomodulator is a IL 18 peptide having more than 80% or 90% or 95% similarity to SEQ ID NOs 44 to 46.

[0301] Clause 52. The tumor homing bacterium of clause 42, wherein the heterologous immunomodulator is a SIRP alpha having more than 80% or 90% or 95% similarity to SEQ ID NOS 37-40.

[0302] Clause 53: The tumor homing bacterium of any one of clauses 34 to 52, wherein the strain are bacteria genetically modified to include a deletion of any one or all of the following genes: stm3120, arginine deiminase (adl), Aminoglycoside (3") (9) adenylyl transferase (aadA), and AAC(6’)-Iaa (aac6) as compared to non- attenuated bacteria of the species Salmonella enterica.

[0303] Clause 54 The tumor homing bacterium of any one of clauses 34 to 52, wherein the inducible prokaryotic expression cassette is inserted to replace the adl, ttrA, or Stm3120 gene.

[0304] Clause 55. The tumor homing bacterium of any one of clauses 34 to 52, wherein the LIGHT peptide is inserted to replace the adl locus.

[0305] Clause 56. The tumor homing bacterium of any one of clauses 34 to 55, wherein one or more inducible prokaryotic expression cassettes comprises a polynucleotide encoding a series of two or three or more heterologous immunomodulators selected from the group consisting of: a SIRP alpha, GMCSF, LIGHT, and IL18, each separated by a ribosomal binding site or cleavage site.

[0306] Clause 57. The tumor homing bacterium of any one of clauses 34 to 56, wherein genetically modified tumor homing bacterium is an attenuated bacterium.

[0307] Clause 58. The tumor homing bacterium of clause 57, wherein the attenuated bacterium is selected from the list consisting of: Salmonella spp., Yersinia spp., Bordetella spp.,Escherichia coli, Shigella spp., Burkholderia mallei, Burkholderia pseudomallei and Pseudomonas aeruginosa.

[0308] Clause 59. The tumor homing bacterium of any one of clauses 57 to 58, wherein the attenuated bacterium is selected from a genus being a Salmonella and / or a Pseudomonas.

[0309] Clause 60. The attenuated bacterium of any one of clauses 557 to 59, wherein the attenuated bacterium is a Salmonella Typhimurium.

[0310] Clause 61. The tumor homing bacterium of any one of clauses 34 to 60, wherein the expression cassettes are in an auto replicative vector or plasmid.

[0311] Clause 62. The tumor homing bacterium of any one of clauses 34 to 60, wherein each of the prokaryotic expression cassettes are chromosomally integrated.

[0312] Clause 63. Tumor homing bacterium, genetically modified for inducible tumor-local delivery of multiple heterologous immunomodulators, said tumor homing bacterium being attenuated bacteria having no deactivating mutations in an outer membrane and having a total of two or more prokaryotic expression cassettes comprising: i. one or more constitutive prokaryotic expression cassettes comprising two or more homologous neoantigen polynucleotides, each associated with a transport signal from a distinct transport system, ii. an aspirin inducible prokaryotic expression cassette encoding an immunomodulator fusion peptide comprising a heterologous immunomodulator associated with a secretion signal, and iii. a regulating expression cassette encoding a regulator positioned in a trans configuration relative to the aspirin-inducible expression cassette and / or inserted at an aadA locus (i.e., replacing the aadA gene).

[0313] Clause 64. The tumor homing bacterium of clause 63, wherein the heterologous immunomodulator is selected from a list consisting of: Interleukin- 18 (IL- 18), Tumor Necrosis Factor Superfamily Member 14 (LIGHT), Signal Regulatory Protein Alpha (SIRPa), CD40 Ligand (CD40L), C-C Motif Chemokine Ligand 5 (CCL5), Anti-ILIORI peptide, Granulocytemacrophage colony stimulating factor (GM-CSF), C-C Motif Chemokine Ligand 21 (CCL21), Short salmonella flagellin B (fliC) and DacA.

[0314] Clause 65. The tumor homing bacterium of clause 63, wherein the heterologous immunomodulator is selected from a list consisting of: LIGHT, GMCSF, SIRP alpha, and IL 18, wherein the inducible promoter is induced by a small molecule that is safe to administer to a subject.

[0315] Clause 66. The tumor homing bacterium of any one of clauses 63 to 65, wherein the secretion signal is from a Type III secretion system or an inner membrane secretion system (e.g., sec or tat secretion system).

[0316] Clause 67. The tumor homing bacterium of clause 66, wherein the secretion signal is from a Type III secretion system.

[0317] Clause 68. The tumor homing bacterium of clause 66, wherein the secretion signal is from a sec or tat secretion system.

[0318] Clause 69. The tumor homing bacterium of any one of clauses 63 to 668, wherein the transport signals from distinct transport systems are from a Type III and a Type V secretion system.

[0319] Clause 70. The tumor homing bacterium of any one of clauses 63 to 68, wherein the transport signals are from a surface display system, a Type III secretion system as well as a Type V secretion system.

[0320] Clause 71. The tumor homing bacterium of any one of clauses 63 to 70, wherein upon induction, the heterologous immunomodulator peptide is secreted and / or expressed in a biologically active form.

[0321] Clause 72. The tumor homing bacterium of any one of clauses 63 to 71, wherein induction of the tumor homing bacterium by a single and safe dose of aspirin administered to a subject, provides a tumor reducing dose of heterologous immunomodulators.

[0322] Clause 73. The tumor homing bacterium of any one of clauses 63 to 71 , wherein the tumor homing bacterium is adapted to provide a therapeutic effect on a tumor upon at least partial tumor colonization in a subject and administration of a safe dose of a salicylic acid or derivative thereof.

[0323] Clause 74. The tumor homing bacterium of any one of clauses 63 to 73, wherein a ratio of the number of inducible prokaryotic expression cassettes to the regulating expression cassette is 2: 1 or more.

[0324] Clause 75. The tumor homing bacterium of any one of clauses 63 to 74, wherein the regulating expression cassette encoding a regulator is in trans position relative to the one or more inducible prokaryotic expression cassettes.

[0325] Clause 76. The tumor homing bacterium of any one of clauses 63 to 75, wherein the heterologous immunomodulator is selected from the group consisting of: a SIRP alpha, GMCSF, LIGHT, and IL 18.

[0326] Clause 77. The tumor homing bacterium of any one of clauses 63 to 76, wherein the inducible prokaryotic expression cassette is a polycistronic vector comprising two or more, three or more or four or more polynucleotide sequences selected from the list of: LIGHT, SIRP alpha, GMCSF and IL 18.

[0327] Clause 78. The tumor homing bacterium of any one of clauses 65 and 76, wherein the heterologous immunomodulator peptide is a LIGHT peptide.

[0328] Clause 79. The tumor homing bacterium of 65 and 76, wherein the heterologous immunomodulator is a GMCSF peptide.

[0329] Clause 80. The tumor homing bacterium of any one of clauses 65 and 76, wherein the heterologous immunomodulator is a IL 18 peptide.

[0330] Clause 81. The tumor homing bacterium of any one of clauses 65 and 76, wherein the heterologous immunomodulator is a SIRP alpha.

[0331] Clause 82. The tumor homing bacterium bacteria of any one of clause 63 to 81, wherein the tumor homing bacterium is modified to include a deletion of any one or all of the genes: stm3120, arginine deiminase (adl), Aminoglycoside (3") (9) adenylyl transferase (aadA), and AAC(6’)-Iaa (aac6) as compared to non-attenuated bacteria of the species Salmonella enterica.

[0332] Clause 83. The tumor homing bacterium of any one of any one of clauses 63 to 82, wherein the inducible prokaryotic expression cassette is inserted at the adl, ttrA, or Stm3120 locus.

[0333] Clause 85. The tumor homing bacterium of any one of clauses 65 to 78 and 82, wherein the LIGHT peptide is inserted at the adl locus.

[0334] Clause 86. The tumor homing bacterium of any one of clauses 63 to 85, wherein one or more inducible prokaryotic expression cassettes comprises a polynucleotide encoding two or three or more heterologous immunomodulator selected from the group consisting of: a SIRP alpha, GMCSF, LIGHT, and IL 18.

[0335] Clause 87. The tumor homing bacterium of any one of clauses 63 to 86, wherein the tumor homing bacterium is an attenuated bacterium.

[0336] Clause 88. The tumor homing bacterium of clause 87, wherein the attenuated bacteria is selected from the bacteriums consisting of: Salmonella spp., Yersinia spp., Bordetella spp., Escherichia coli, Shigella spp., Burkholderia mallei, Burkholderia pseudomallei and Pseudomonas aeruginosa.

[0337] Clause 89. The tumor homing bacterium of any one of clauses 87 to 88, wherein the bacterium is selected from a genus being a Salmonella and / or a Pseudomonas.

[0338] Clause 90. The bacterium of any one of clauses 87 to 89, wherein the bacterium is a Salmonella Typhimurium.

[0339] Clause 91. The tumor homing bacterium of any one of clauses 63 to 90, wherein the expression cassettes are in an auto replicative vector or plasmid.

[0340] Clause 92. The tumor homing bacterium of any one of clauses 63 to 90, wherein the expression cassettes are chromosomally integrated.

[0341] Clause 93. A vaccine comprising a tumor homing bacterium according to any one of clauses 1 to 92 and one or more pharmaceutically acceptable carriers or excipients.

[0342] Clause 94. A method of treating a cancer of a subject in need thereof comprising: i. administering to the subject an effective amount of the vaccine of clause 93; upon tumorcolonization of the tumor homing bacterium in a cancer tumor and / or a reduction in a systemic bacterial load, administering an inducer.

[0343] Clause 95. The method according to clause 94, wherein treatment of a cancer comprises tumor reduction.

[0344] Clause 96. The method according to any one of clauses 94 or 95, wherein administering a safe dose of aspirin results in a tumor reducing dose of three or more immunomodulators selected from the list consisting of: LIGHT, GMCSF, SIRP alpha, and IL 18.

[0345] Clause 97. The method according to any one of clauses 94 to 96, wherein the tumor homing bacterium is attenuated Gram negative bacterium and is further genetically modified to constitutively express multiple cancer associated antigens (e.g., neoantigens) fusion peptides, wherein homologous neoantigens are secreted by distinct pathways.

[0346] Clause 98. The method according to any one of clauses 94 to 97, wherein the tumor homing bacterium is genetically modified to co-express two homologous cancer associated antigens (e.g., neoantigens), each respective cancer associated antigens fused to a transport signal from a distinct bacterial transport system.

[0347] Clause 99. The method according to any one of clauses 94 to 98, wherein the inducer is a salicylate or salicylic acid.

[0348] Clause 100. The method according to any one of clauses 94 to 99, wherein administering an inducer is by periodic administration, such as readministering a salicylate or salicylic acid after a pause in expression or secretion of a heterologous immunomodulator.

[0349] Clause 101. The method of clause 99, wherein administering an inducer is readministering a salicylate or salicylic acid after a pause in expression or secretion of an immunomodulator.

[0350] Clause 102. The method of clause 100, wherein administering an inducer is after day 18, post tumor homing bacterium administration.

[0351] Clause 103. The method of any one of clauses 94 to 102, wherein administering to the subject provides systemic exposure to the vaccine in a first stage.

[0352] Clause 104. The method of any one of clauses 94 to 103, wherein administering to the subject is by parenteral administration.

[0353] Clause 105. The method of any one of clauses 94 to 103, wherein administering to the subject is by oral administration.

[0354] Clause 106. The method of any one of clauses 94 to 105, wherein the cancer is selected from the group consisting of breast, melanoma, colorectal cancer, lung cancer, gastric cancer, pancreatic cancer, ovarian cancer, bone cancer and brain cancer.

[0355] Clause 107. A method of treating a cancer in a subject in need thereof comprising: i. administering a Gram-negative tumor homing bacterium to a subject in need thereof, said bacterium genetically modified for aspirin inducible expression of a immunomodulator fusion peptide, said immunomodulator fusion peptide comprising a heterologous immunomodulator characterized by a 1 to 4% cysteine content and having a length of between 30 to 400 amino acids and being linked to an inner membrane secretion signal, such as a Sec or tat secretion system; upon tumor-colonization of the bacteria in a cancer tumor and / or reduction of a systemic bacterial load in a subject, administering to the subject a safe dose of aspirin.

[0356] Clause 108. A method of treating a cancer in a subject in need thereof comprising: i. administering a Gram-negative tumor homing bacterium to a subject in need thereof, said Gram negative bacteria genetically modified for aspirin inducible expression of immunomodulator fusion peptide, said immunomodulator fusion peptide comprising an IL- 18 linked to a Type IIIsecretion signal; upon tumor-colonization of the bacteria in a cancer tumor and / or reduction of a systemic bacterial load in a subject, administering to the subject a safe dose of aspirin.

[0357] Clause 109. The method of any one of clauses 107 or 108, wherein administering is systemically administering, such as by intravenous route.

[0358] Clause 110. The method of any one of clauses 107 to 108, wherein treating a cancer is providing a measurable reduction in tumor volume.

[0359] Clause 111. The method of any one of clause 107 to 110, wherein the Gram-negative tumor homing bacterium is selected from a list consisting of: Salmonella spp., Yersinia spp., Bordetella spp., Escherichia coli, Shigella spp., Burkholderia mallei, Burkholderia pseudomallei and Pseudomonas aeruginosa and preferably Salmonella.

[0360] Clause 112. The method of clause 107, wherein the heterologous immunomodulator is a LIGHT, GMCSF, or SIRP alpha peptide.

[0361] Clause 113. The method of any one of clauses 107 to 112, wherein administering to the subject a safe dose of aspirin results in the presence (e.g., via secretion) of a tumor reducing dose of biologically active heterologous immunomodulator in the tumor environment.

[0362] Clause 114. A vaccine of clause 93 for use in the treatment of cancer in a subject, wherein the vaccine is administered to a subject in need and wherein upon tumor-colonization of the tumor homing bacterium in a cancer tumor and / or a reduction in a systemic bacterial load an inducer is administered.

[0363] Clause 115. The vaccine for use of clause 114, wherein treatment of a cancer comprises tumor reduction.

[0364] Clause 116. The vaccine for use to any one of clauses 114 or 115, wherein administration of a safe dose of aspirin results in a tumor reducing dose of three or more immunomodulators selected from the list consisting of: LIGHT, GMCSF, SIRP alpha, and IL 18.

[0365] Clause 117. The vaccine for use according to any one of clauses 114 to 116, wherein the tumor homing bacterium is attenuated Gram negative bacterium and is further genetically modified to constitutively express multiple cancer associated antigens (e.g., neoantigens) fusion peptides, wherein homologous neoantigens are secreted by distinct pathways.

[0366] Clause 118. The vaccine for use according to any one of clauses 114 to 117, wherein the tumor homing bacterium is genetically modified to co-express two homologous cancer associatedantigens (e.g., neoantigens), each respective cancer associated antigens fused to a transport signal from a distinct bacterial transport system.

[0367] Clause 119. The vaccine for use according to any one of clauses 114 to 118, wherein the inducer is a salicylate or salicylic acid.

[0368] Clause 120. The vaccine for use according to any one of clauses 114 to 119, wherein an inducer is administered by periodic administration, such as readministration of a salicylate or salicylic acid (or derivative thereof) after a pause in expression or secretion of a heterologous immunomodulator.

[0369] Clause 121. The vaccine for use of any one of clauses 114 to 120, wherein administration of an inducer is readministration of a salicylate or salicylic acid after a pause in expression or secretion of an immunomodulator.

[0370] Clause 122. The vaccine for use of any one of clauses 114 to clause 120, wherein administration of an inducer is after day 18, post tumor homing bacterium administration.

[0371] Clause 123. The vaccine for use of any one of clauses 114 to 122, wherein the administration to the subject provides systemic exposure to the vaccine for use in a first stage.

[0372] Clause 124. The vaccine for use of any one of clauses 114 to 123, wherein the vaccine for use is administered to the subject by parenteral administration.

[0373] Clause 125. The vaccine for use of any one of clauses 114 to 123, wherein the vaccine for use is administered to the subject by oral administration.

[0374] Clause 126. The vaccine for use of any one of clauses 114 to 125, wherein the cancer is selected from the group consisting of breast, melanoma, colorectal cancer, lung cancer, gastric cancer, pancreatic cancer, ovarian cancer, bone cancer and brain cancer.EXAMPLES

[0375] Reference is now made to the following examples, which together with the above descriptions illustrate some embodiments of the invention in a non-limiting fashion.

[0376] For all examples, unless otherwise indicated, ASA in vitro is administered at 200pM; ASA is administered in vivo at 25mg / kg, per os and aPD (anti-PDl) in vivo is administered at 150μg , i.p (Intraperitoneal )Example 1 : Kinetics of the aspirin-mediated Induction of Expression with a salR-pSal System in MiceTo characterize the kinetics of the induction and expression decay of genes under aspirin control, a Salmonella Typhimurium STM3120 strain expressing luciferase under the salR-pSal system (STM3120-Luc) was generated and injected into mice. C57BL / 6 mice were injected with 10^5 MC38 cells in the right flank. When tumors reached a volume of -100 mm3, mice were injected with one dose (Day 0) of STM3120-Luc (10^6 CFU, IV). On day 19, after bacteria had colonized the tumors while clearing out of other tissues, mice received a dose of ASA (25 mg / kg, gavage). Mice were imaged for bioluminescence on days 18 (before induction), 20 (1 day following induction), 21 (2 days following induction), and 22 (3 days following induction). In FIG. 5, images of 3 different mice (rows) at 4 different time points (columns, days 18, 20, 21 , and 22, as indicated in the top left corners) are shown. Induction of gene expression was detected 1-day following induction, and the complete decay of expression was observed 3 days following induction. Black and white photos are labeled with a plus sign and double plus sign (more luminescence) to indicate cases where luminescence is present on day 20. Note that the induction period is controlled and limited.

[0377] ASA= acetylsalicylic acid, Aspirin; CFU=colony forming units; IV=intravenousExample 2: Trans configuration of salR regulator and pSal promoter in the bacteria genome improves protein expression.

[0378] Salmonella Typhimurium (STM3120) bacteria were genetically modified to express IL 18 under ASA induction. The IL 18 expression cassette (pSal promoter + IL 18 coding sequence) was inserted into the bacterial genome at the aac6 locus (left panel) or ttrA locus (right panel). The SalR regulator expression cassette was either inserted together with the IL 18 expression cassette (cis) or into a distant locus on the genome (trans). To evaluate the ASA mediated induction of IL 18, bacteria were diluted from an overnight starter and grown to OD600of 1.0 with (+) or without (-) ASA (200pM) and lysates were subjected to Western blot. As presented in the Western blot of FIG. 6, where ASA mediated induction of IL18 is shown for both loci, bacteria with the SalR regulator cassette in trans showed a more efficacious induction of the expression. EFTU is a bacterial elongation factor (eEFlAl) used as positive control. Salmonella Typhimurium (STM3120) bacteria were genetically modified to express IL 18 under ASA induction. The IL 18 expression cassette (pSal promoter + IL 18 coding sequence) wasinserted into the bacterial genome at the aac6 locus (A) or ttrA locus (B). The SalR regulator expression cassette was either inserted together with the IL 18 expression cassette (cis) or into a distant locus on the genome (trans). To evaluate the ASA mediated induction of IL18, bacteria were diluted from an overnight starter and grown to OD600of 1.0 with (+) or without (-) ASA and lysates were subjected to Western blot. As presented in the Western blot of FIG. 6, where ASA mediated induction of IL 18 is shown for both loci, bacteria with the SalR regulator cassette in trans showed a more efficacious induction of the expression. EFTU is a bacterial elongation factor (eEFlAl) used as positive control.Example 3: Validation of functionality of IL8 secreted from bacteria.

[0379] Salmonella typhimurium STM3120 mutant transformed with a pQE plasmid expressing hIL18 or unrelated protein (LIGHT) following ASA induction, were diluted from an overnight starter and grown in LB and ASA (200microM) to OD600of 0.8-1. Bacterial cultures were pelleted and the supernatant was filtered through 0.22μm filters. Twenty pl of bacterial supernatant was mixed with IL18 responsive HEK Blue cells (180 microliter containing 5*104cells / well) and incubated for at least 20 hours at 5% CO2, 37°C incubator. Medium was then mixed with a Quanti-blue reagent and OD620nm was measured using Cytation 3 platereader (Biotek) to determine IL 18 activity. Graph in FIG. 7 represents data from 4 different experiments with 3 technical repeats in each (total of 12). Each experiment included LB only as negative control (not shown) and recombinant IL 18 as positive control and each value was normalized to the average induction values of recombinant hIL18 (50 pg / ml) in the respective experiment. The Graph in FIG. 7 shows functional induction of IL 18 HEK blue cells by the supernatants of bacteria expressing and secreting IL 18 as compared to bacteria expressing and secreting an unrelated payload (LIGHT, Bact. + Unrel.). Statistical analysis was done on Graphpad, Mann Whitney test, * - p value <0.05)Example 4: Validation of functionality of LIGHT, secreted from bacteria.

[0380] Salmonella typhimurium (STM) 3120 mutant transformed with a pQE plasmid expressing LIGHT following ASA induction or unrelated protein (IL 18), were diluted from an overnight starter and grown in LB and ASA (200microM) to OD600of 0.5. Bacterial cultures were pelleted and the supernatant was filtered through 0.22μm filters. Ten pl of bacterial supernatant was added to A375 human melanoma cells (plated at 2 x 10^5 cells per well in 6- well plates the day before) and incubated for 3h at 5% CO2, 37°C incubator. RNA was isolatedfrom the cells to evaluate the expected induction of CXCL8 (IL8) following addition of LIGHT. Following RNA isolation and reverse transcription, the cDNA was subjected to qPCR to assess gene expression of, ACTB, and CXCL8 (IL8). Fold change in gene expression was determined using the AACt method, calculating ACt of IL8 vs ACTB for both conditions (bacteria with LIGHT and bacteria with unrelated) and then ACt between both conditions (2ΔΔCt). The graph in FIG. 8 shows functional induction and secretion of LIGHT. Statistical analysis was done on Graphpad, Wilcoxon signed-rank test, one-tailed , * - p value <0.05.Example 5: Validation of functionality of GM-CSF, secreted from bacteria.

[0381] Salmonella typhimurium STM3120 mutant engineered to express (episomal or genomic) human GM-CSF or an unrelated payload (LIGHT), under an ASA inducible system, were diluted from an overnight starter and grown in LB and ASA (200 microM) to OD600of 1.0. Bacterial cultures were pelleted and the supernatant was filtered through 0.22μm filters. Forty pl of bacterial supernatant was added to 40 pl iLite® GM-CSF luciferase reporter cells (SVAR) (prepared and used according to the manufacturer instructions ) and incubated for 5h at 5% CO2, 37°C incubator. Luminescence of luciferase was then developed and measured using the Dual- Glo® Luciferase Assay System (Promega) and Cytation 3 platereader (Biotek). Graph in FIG. 9 represents data from 4 different experiments. Experiments included LB + ASA only as negative control (No) and recombinant GM-CSF (rGM-CSF, 200pg / ml) as positive control. The graph in FIG. 9 shows functional induction of GM-CSF reporter cells following incubation with supernatants of bacteria expressing and secreting GM-CSF (Bact+GM-CSF) as compared to bacteria expressing and secreting an unrelated payload (Bact + Unrel.). Statistical analysis was done on Graphpad, Mann Whitney test, * - p value <0.05)Example 6:Validation of functionality of SIRPa, expressed and secreted by bacteria.

[0382] (A) Salmonella typhimurium STM3120 mutant transformed with a pQE plasmid expressing extracellular domain of human SIRPa or unrelated protein (LIGHT), were diluted from an overnight starter and grown in LB and ASA (200microM) for payloads induction to OD600of 0.9. Bacterial cultures were pelleted, and pellets were resuspended in B-PER (ThermoFisher, cat. 78443) at a ratio of 4ul buffer per Img pellet. Next, 50,000 Jurkat cells (CD47+, SIRPa-) were cocultured with 2.5mg suspended pellet of SIRPa or LIGHT expressing bacteria or different concentrations of recombinant human SIRPa in a total volume of lOOmicroliter RPMI medium supplemented with 10% FCS, glutamine, pen / strep and pyruvate.After Ih of incubation, cells were washed and stained for 30min with 1 pg / ml anti-human SIRPa- APC (abeam, cat. Ab275641). Samples were acquired by Beckman Coulter Life Sciences CytoFLEX. Binding of bacterial-expressed SIRPa to its receptor (CD47) on Jurkat cells was demonstrated by Jurkat cells that were stained positive for SIRPa. The binding of SIRPa was expressed by the Geometric mean of the SIRPa signal and is presented in the graph in FIG. 10A.

[0383] (B) To evaluate the secretion of SIRPa, bacterial supernatants were diluted from overnight cultures and grown in 2 ml cultures with (+) or without (-) ASA (200 pM) to OD6001. Bacteria were pelleted and supernatants were filtered (0.22 micrometer pore size), protein was concentrated with protein precipitation kit (A&A Biotechnology) according to the manual. All the resulting protein was loaded on the gel and subjected to western blot. FIG. 10B shows the detection of SIRPa protein (detected with an anti-Flag Ab, Flag tag was added to C-terminal end of SIRPa) in the secreted fraction of bacteria.Example 7: Validation of expression of Neoantigens and payloads in Bacteria.

[0384] Salmonella Typhimurium (STM3120) bacteria were genetically modified to introduce the MC38 specific neoantigen ADPGK in 4 copies, each with its presentation mode (ompA- ADPGK : cell wall attached, T3SS ADPGK, T5SS ADPGK and T2SS : Sec-mediated secretion). The bacteria was further modified to express 4 different immune modulating payloads (SIRPa, GM-CSF, IL 18 and LIGHT) under the control of Aspirin. To evaluate the expression of the neoantigens and the ASA mediated induction of the payloads, bacteria were diluted from an overnight starter and grown to OD600of 1.0 with (+) or without (-) ASA (200 pM) and lysates were subjected to Western blot. Figure shows the successful insertion of multiple modifications and expression of all inserted coding sequences. EFTU is a bacterial elongation factor (eEFlAl) used as positive control.Example 8: Immune modulating payload (IMP) mediated changes in mice Tumors and Tumor Draining Lymph Nodes (LN) immune profile.

[0385] C57BL / 6 mice were injected with 10^5 MC38 cells in the right flank. When tumors reached sizes of -100 mm3, mice were injected IV with 106bacteria (attenuated Salmonella Typhimurium) expressing Neoantigens only (Nag) or bacteria expressing neoantigens and one payload : IL18 (FIG. 12), LIGHT (FIG. 13), GM-CSF (FIG. 14), and SIRPa (FIG. 15). Each strain expresses one immunomodulator to evaluate the specific effect of the tested immunomodulator. Mice received ASA (25mg / kg, per os) on days 3 and 7 post bacterial injectionto induce expression of payloads and aPDl (anti-PDl, 150microgram, i.p) on day 3. Mice were sacrificed on day 10 and tumors and tumor draining lymph nodes were subjected to immune profiling by FACS analysis. Immune populations were defined as follows: Macrophages were identified as CD45+CD1 lb+F4 / 80+, the markers I-A / I-E and CD206 were used to distinguish between Ml (I-A / I-E+ CD206-) and M2 (CD206+) macrophages. Neutrophils were identified as CD45+CD1 lb+Ly6G+, Mast cells were identified as CD45+CD3-cKit+, inflammatory monocytes were identified as CD45+CD1 lb+F4 / 80-Ly6ChiLy6G-, cDCl (Conventional type 1 dendritic cells) were identified as CD45+CD1 lb+F4 / 80-CDl lc+MHCII+XCRl+, and cDC2 (Conventional type 2 dendritic cells) as CD45+CD1 lb+F4 / 80-CDl lc+MHCII+XCRl-CD172+. CD4 T cells as CD45+CD3+ TCRab+TCRγδ-CD4+, CD8 T cells as CD45+CD3+TCRab+TCRγδ-CD8+, and memory cells were identified with CD62L+CD44+ in the CD4 or CD8 populations. Antigen(ADPGK)-specific CD8 T cells were identified with CD45+CD3+CD8+ tetramer+(ADPGK specific tetramers ). Statistical data was analyzed with the GraphPad Prism9.5.1 software, p values were calculated by the Mann-Whitney test, with values < 0.05 being considered significant.Example 9: Evaluation of the effect of bacteria expressing various immunomodulators in the TME on tumor progression in mice.

[0386] C57BL / 6 mice were injected with 10^5 MC38 cells in the right flank. When tumors reached a volume of ~100mmA3, mice received intravenous (i.v.) injections with 10^6 CFU of Salmonella bacteria expressing a specific payload, or background bacteria (adl KO) that had a deletion of adl gene, the locus replaced with the immunomodulators. Immunomodulator expression was induced at day 3 post bacterial injection by aspirin administration per os (25mg / kg), given twice a week. Mice received weekly administration of 150microgram anti-PDl, i.p. The graph in FIG. 16 shows the average size of tumors (bars) and distribution of tumor sizes for the different immunomodulator treatments as measured on day 17 following bacterial injection. Statistical analysis was done on Graphpad, Mann Whitney test, * - p value <0.05)Example 10: Efficacy of treatment of mice with bacteria expressing one or multiple payloads.

[0387] C57BL / 6 mice were injected with 105MC38 colon cancer cells in the right flank. When tumors reached a volume of -100 mm3, mice received intravenous (IV) injections of 106CFU ofthe indicated strains or PBS as control. Mice received weekly administration of 150microgram anti-PDl, i.p. (“PBS only” group was not treated) and ASA on day 3 post bacterial injection per os (25mg / kg), given twice a week. Graphs show the growth of the tumors over time. Treatment groups: “PBS only” received PBS instead of bacterial injection and did not receive aPDl treatment, “aPDl + PBS” received PBS instead of bacterial injection and received aPDl treatment, “aPDl + BactNeo” received bacteria expressing neoantigen only and aPDl , “aPDl + BactNeo+GM-csr” received bacteria expressing neoantigen together with GM-CSF and aPD, “aPDl + BactNeo+siRPa” received bacteria expressing neoantigen together with SIRPa and aPDl, “aPDl + BactNeo+LiGHT” received bacteria expressing neoantigen together with LIGHT and aPDl , and “aPDl + BactNeo+siRP+GM+LiGHT” received bacteria expressing neoantigen together with the combination of GM-CSF, SIRPa and LIGHT, and aPDl. In FIG. 17, a clear increase in efficacy can be observed when all 3 payloads are expressed in the bacteria.REFERENCES

[0388] Henikoff S, Henikoff JG. Amino acid substitution matrices from protein blocks. Proc Natl Acad Sci U S A. 1992 Nov 15;89(22): 10915-9. doi: 10.1073 / pnas.89.22.10915. PMID: 1438297; PMCID: PMC50453.

[0389] Arrach N, Cheng P, Zhao M, Santiviago CA, Hoffman RM, McClelland M. High- throughput screening for salmonella avirulent mutants that retain targeting of solid tumors. Cancer Res. 2010 Mar 15;70(6):2165-70. doi: 10.1158 / 0008-5472.CAN-09-4005. PMID: 20231149; PMCID: PMC4103738.

[0390] Lloyd, S.A., Sjostrom, M., Andersson, S. and Wolf-Watz, H. (2002), Molecular characterization of type III secretion signals via analysis of synthetic N-terminal amino acid sequences. Molecular Microbiology, 43: 51-59. https: / / doi.Org / 10.1046 / j.1365-2958.2002.02738.

Claims

CLAIMSWhat is claimed is:

1. A tumor-homing bacterium genetically modified for inducible tumor-local delivery of multiple heterologous immunomodulators, said tumor-homing bacterium being an attenuated Gramnegative bacterium comprising three prokaryotic expression cassettes comprising: i. one or more constitutive prokaryotic expression cassettes comprising two or more homologous cancer associated antigens, each of the respective homologous cancer associated antigens associated with a transport signal from a distinct transport system, ii. a regulating expression cassette encoding a regulator; and iii. one or more aspirin-inducible prokaryotic expression cassettes encoding a set of immunomodulator fusion peptides operably linked to an inducible promoter, each of said immunomodulator fusion peptides comprising a heterologous immunomodulator associated with a secretion signal, said heterologous immunomodulator selected from a list consisting of: LIGHT, GMCSF, SIRP alpha, and IL18.

2. The tumor-homing bacterium of claim 1, wherein the aspirin-inducible prokaryotic expression cassettes encodes three peptides selected from the list of: LIGHT, SIRP alpha, GMCSF and IL18.

3. The tumor-homing bacterium of claim 1 or claim 2, wherein a ratio of a number of inducible prokaryotic expression cassettes to the regulating expression cassette is 2:1 or more.

4. The tumor-homing bacterium of any one of claims 1 to 3, wherein upon induction, the immunomodulator is secreted in a biologically active form.

5. The tumor-homing bacterium of any one claims 1 to 4, wherein the tumor-homing bacterium has no deactivating mutation in an outer membrane protein; and wherein each of the respective heterologous immunomodulators are linked to a secretion signal from a Type III secretion system or an inner membrane secretion system.

6. The tumor-homing bacterium of claim 5 wherein the heterologous immunomodulator has a cysteine content of more than 1% and wherein each of the respective heterologous immunomodulators is linked to the secretion signal from a sec or tat secretion system.

7. The tumor-homing bacterium of any of claims 2 to 6, wherein a polynucleotide encoding the LIGHT peptide is inserted at the adl locus.

8. The tumor-homing bacterium of any of claims 1 to 7, wherein the distinct transport signals are a Type III and a Type V secretion signal.

9. The tumor-homing bacterium of claim 8, wherein the distinct transport signals are each of a surface display signal, a Type III secretion signal and a Type V secretion signal.

10. The tumor-homing bacterium of any of claims 1 to 9, wherein the aspirin-inducible prokaryotic expression cassette is inserted at an adl, ttrA, or Stm3120 locus.

11. The tumor-homing bacterium of any of claims 1 to 10, wherein the regulating expression cassette is in trans position relative to the aspirin-inducible prokaryotic expression cassette.

12. The tumor-homing bacterium of any one of claims 1 to 11, wherein the regulating expression cassette encoding a regulator is either: i. inserted at a aadA locus rendering a deactivating mutation in the aadA gene; or ii. is in a trans position relative to the aspirin-inducible prokaryotic expression cassette and wherein the aspirin-inducible prokaryotic expression cassette is induced by a salicylic acid , salicylate or derivative thereof.

13. The tumor-homing bacterium of any one of claims 1 to 12, wherein the aspirin-inducible prokaryotic expression cassette is a polycistronic vector comprising three or more polynucleotide sequences selected from the list of: LIGHT, SIRP alpha, GMCSF and IL18.

14. The tumor-homing bacterium of any one of claims 1 to 13, wherein the homologous cancer associated antigens are homologous neoantigens.

15. The tumor-homing bacterium of any of claims 1 to 14, wherein the tumor-homing bacterium is selected from the list consisting of: Salmonella spp., Yersinia spp., Bordetella spp., Escherichia coli, Shigella spp., Burkholderia mallei, Burkholderia pseudomallei and Pseudomonas aeruginosa.

16. The tumor-homing bacterium of claim 15, wherein the bacterium is selected from a genus being a Salmonella and / or a Pseudomonas.

17. The tumor-homing bacterium of claim 16, wherein the bacterium is a species being Salmonella Typhimurium.

18. The tumor-homing bacterium any one of claims 1 to 17, wherein the expression cassettes are chromosomally integrated.

19. A tumor-homing bacterium, genetically modified for inducible tumor-local delivery of multiple heterologous immunomodulators, said tumor-homing bacterium being attenuated bacteria having no deactivating mutations in an outer membrane and having a total of two prokaryotic expression cassette comprising: i. one or more constitutive prokaryotic expression cassettes comprising two or more homologous neoantigen polynucleotides, each associated with a transport signal from a distinct transport system; ii. an inducible prokaryotic expression cassette encoding an immunomodulator fusion peptide comprising a heterologous immunomodulator linked to a secretion signal, and wherein either: a. the secretion signal is an inner membrane secretion signal or a Type III secretion signal; or b. further comprising a prokaryotic expression cassette comprising a regulator positioned upstream or in a trans configuration relative to the inducible prokaryotic expression cassette.

20. The tumor-homing bacterium of claim 19, wherein the inducer is a small molecule selected from a list consisting of L-arabinose, IPTG, a salicylic acid, acetylsalicylic acid or derivative thereof.

21. The tumor-homing bacterium of claim 19 or 20, wherein the inducible prokaryotic expression cassette is an aspirin-inducible prokaryotic expression cassette.

22. A tumor-homing bacterium, genetically modified for inducible tumor-local delivery of multiple heterologous immunomodulators, said tumor-homing bacterium being an attenuated bacterium having no deactivating mutations in an outer membrane and having a total of three prokaryotic expression cassettes comprising: i. one or more constitutive prokaryotic expression cassettes comprising two or more homologous neoantigen polynucleotides, each associated with a transport signal from a distinct transport system,ii. an aspirin inducible prokaryotic expression cassette encoding an immunomodulator fusion peptide comprising a secretion signal linked to a heterologous immunomodulator; iii. a regulating expression cassette inserted at a aadA locus or positioned in a trans configuration relative to an aspirin-inducible expression cassette.

23. A vaccine comprising a population of a bacterium according to any one of claims 1 to 21 and one or more pharmaceutically acceptable carriers or excipients.

24. Use of the vaccine in claim 23, in the treatment of cancer in a subject in need thereof comprising: i. administering to the subject an effective amount of the vaccine; ii. upon tumor-colonization of the bacteria in a cancer tumor and / or a reduction in a systemic bacterial load in a subject; iii. administering to the subject a salicylate, salicylic acid or derivative thereof; and iv. administering to the subject an immune checkpoint modulator.

25. The method of claim 24, wherein administering an inducer is a single-time systemic administration.

26. The method of claim 24 or claim 25, wherein administering an inducer is a periodic administration.

27. The method of any one of claims 23 to claim 26, wherein administering an effective amount of the vaccine is by parenteral administration.

28. The method of any one of claims 23 to claim 27, wherein the cancer is selected from the group consisting of breast, melanoma, colorectal cancer, lung cancer, gastric cancer, pancreatic cancer, ovarian cancer, bone cancer and brain cancer.

29. A method of treating a cancer in a subject comprising: i. administering to a blood stream of a subject in need thereof, a tumor-homing bacterium genetically modified for constitutive multi-model secretion of two homologous cancer associated antigens and inducible expression of a set of heterologous immunomodulators comprising three or more peptides from the following: SIRP alpha, GMCSF, IL- 18 and LIGHT; ii. upon tumor-colonization of the bacteria in a cancer tumor and / or a reduction in a systemic bacterial load, administering an inducer; andiii. administering an immune checkpoint modulator.

30. The method of claim 29, wherein the two homologous cancer associated antigens are two homologous neoantigens.

31. The method of claim 29 or claim 30, wherein administering the tumor-homing bacterium to a blood stream of a subject comprises intravenous or intra-tumoral administration.

32. The method of claim 31, wherein administering to a blood stream of a subject comprises intravenous administration.

33. The method of any one of claims 30 to 32, wherein multi-model secretion of homologous cancer associated antigens comprise secretion by a Type III and a Type V secretion system.

34. The method of claim 33, wherein further to the multi-model secretion, a homologous neoantigen is displayed on the cell wall surface.

35. The method of any one of claims 29 to 35, wherein the set of heterologous immunomodulators comprising all four of the following: SIRP alpha, GMCSF, IL- 18 and LIGHT.

36. The method of any of claims 1 to 34, wherein the tumor-homing bacterium is a Gram-negative bacterium.

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